t4 dna ligase buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 dna ligase buffer
    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new <t>T4</t> DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo"

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx533

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    Figure Legend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Techniques Used: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.
    Figure Legend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Techniques Used: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification

    2) Product Images from "Efficient assembly of very short oligonucleotides using T4 DNA Ligase"

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-291

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Figure Legend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Techniques Used: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).
    Figure Legend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Techniques Used: Ligation, Labeling

    3) Product Images from "Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks"

    Article Title: Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks

    Journal: bioRxiv

    doi: 10.1101/300962

    The Cas9-DSB complex precludes DNA repair activities in vitro. A, Agarose gel electrophoresis of linear dsDNA cut by Cas9, then treated with Proteinase K. B, E. coli colony formation from circular plasmid DNA undergoing the indicated digestion and ligation conditions. Cas9 was denatured at 75C for 10m before addition of phage T4 DNA ligase. Values represent mean +/- s.d., n=3. C, Agarose gel displaying plasmid DNA digested with PmeI restriction endonuclease or Cas9 before incubation with phage T7 exonuclease. D, Schematic depicting the experiment in E testing if Ku70/80 can displace Cas9 from a DSB. The Cas9-DSB complex is formed on an immobilized, fluorescent substrate then challenged with purified Ku70/80. Disruption of the Cas9-DSB complex causes release of the fluorescent DNA end into the soluble fraction. E, Release of fluorescently labeled DNA ends into the soluble fraction after challenging the immobilized target DNA with indicated conditions. NcoI is a restriction endonuclease that cuts the DNA substrate. Values represent mean +/- s.d., n=3.
    Figure Legend Snippet: The Cas9-DSB complex precludes DNA repair activities in vitro. A, Agarose gel electrophoresis of linear dsDNA cut by Cas9, then treated with Proteinase K. B, E. coli colony formation from circular plasmid DNA undergoing the indicated digestion and ligation conditions. Cas9 was denatured at 75C for 10m before addition of phage T4 DNA ligase. Values represent mean +/- s.d., n=3. C, Agarose gel displaying plasmid DNA digested with PmeI restriction endonuclease or Cas9 before incubation with phage T7 exonuclease. D, Schematic depicting the experiment in E testing if Ku70/80 can displace Cas9 from a DSB. The Cas9-DSB complex is formed on an immobilized, fluorescent substrate then challenged with purified Ku70/80. Disruption of the Cas9-DSB complex causes release of the fluorescent DNA end into the soluble fraction. E, Release of fluorescently labeled DNA ends into the soluble fraction after challenging the immobilized target DNA with indicated conditions. NcoI is a restriction endonuclease that cuts the DNA substrate. Values represent mean +/- s.d., n=3.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Plasmid Preparation, Ligation, Incubation, Purification, Labeling

    4) Product Images from "Demonstration of a universal surface DNA computer"

    Article Title: Demonstration of a universal surface DNA computer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh635

    Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.
    Figure Legend Snippet: Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.

    Techniques Used: Ligation

    5) Product Images from "Efficient Modification and Preparation of Circular DNA for Expression in Cell Culture"

    Article Title: Efficient Modification and Preparation of Circular DNA for Expression in Cell Culture

    Journal: bioRxiv

    doi: 10.1101/2022.06.28.497995

    Protocol steps: ( a ) Pipette dsDNA template with T4 DNA ligase and type IIS restriction enzyme reagents, then program incubation at 37 o C (or longer, if desired), followed by the heat activation of T4 DNA ligase at 65 o C for 15 minutes. If left unattended, set the program cycler to pause at 4 o C. ( b ) Add an equal volume of T5 exonuclease reagents to the same tube and digest at 37 o C for 1 hour to 1.5 hours. ( c ) Purify the circularized DNA expression construct from the reaction mix using a DNA cleanup kit.
    Figure Legend Snippet: Protocol steps: ( a ) Pipette dsDNA template with T4 DNA ligase and type IIS restriction enzyme reagents, then program incubation at 37 o C (or longer, if desired), followed by the heat activation of T4 DNA ligase at 65 o C for 15 minutes. If left unattended, set the program cycler to pause at 4 o C. ( b ) Add an equal volume of T5 exonuclease reagents to the same tube and digest at 37 o C for 1 hour to 1.5 hours. ( c ) Purify the circularized DNA expression construct from the reaction mix using a DNA cleanup kit.

    Techniques Used: Transferring, Incubation, Activation Assay, Expressing, Construct

    6) Product Images from "Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo"

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx533

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    Figure Legend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Techniques Used: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.
    Figure Legend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Techniques Used: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification

    7) Product Images from "Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination"

    Article Title: Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination

    Journal: Nature Communications

    doi: 10.1038/s41467-021-21275-4

    Characterization of various genomic DNA digestion/DNA assembly combinations in the CAPTURE method. a Schematics of T4 DNA polymerase exo + fill-in DNA assembly. In step 1, DNA molecules ends are chewed back by T4 DNA polymerase to create ssDNA overhangs. The reaction mixture’s temperature is increased to 75 °C to inactivate T4 DNA polymerase and potentially remove ssDNA secondary structures. Temperature is then decreased to 50 °C to allow for ssDNA overhang hybridization. In step 2, by addition of fresh T4 DNA polymerase, and dNTPs, DNA gaps in the hybridized DNA molecule are filled. E. coli DNA ligase is then used to ligate the nicks and produce the final assembly product. b Comparison of different digestion/DNA assembly combinations in cloning four high GC-content BGCs from Actinomycetes. The Fn Cas12a/T4 exo + fill-in strategy showed ~100% cloning efficiency for all four target BGCs. RE: restriction enzymes. For each cloning experiment, at least seven colonies were selected and the purified plasmids from each colony were analyzed by restriction digestion. The cloning efficiencies were calculated as the ratio of correct colonies to the total number of checked colonies. Each experiment was performed in three biological replicates and data are presented as mean values ± standard error (SEM). c Summary of results for cloning uncharacterized BGCs using CAPTURE. BGCs ranging from 10 to 113 kb can be robustly cloned using the CAPTURE method at close to 100% efficiency regardless of their GC-content. Source data are provided as a Source Data file.
    Figure Legend Snippet: Characterization of various genomic DNA digestion/DNA assembly combinations in the CAPTURE method. a Schematics of T4 DNA polymerase exo + fill-in DNA assembly. In step 1, DNA molecules ends are chewed back by T4 DNA polymerase to create ssDNA overhangs. The reaction mixture’s temperature is increased to 75 °C to inactivate T4 DNA polymerase and potentially remove ssDNA secondary structures. Temperature is then decreased to 50 °C to allow for ssDNA overhang hybridization. In step 2, by addition of fresh T4 DNA polymerase, and dNTPs, DNA gaps in the hybridized DNA molecule are filled. E. coli DNA ligase is then used to ligate the nicks and produce the final assembly product. b Comparison of different digestion/DNA assembly combinations in cloning four high GC-content BGCs from Actinomycetes. The Fn Cas12a/T4 exo + fill-in strategy showed ~100% cloning efficiency for all four target BGCs. RE: restriction enzymes. For each cloning experiment, at least seven colonies were selected and the purified plasmids from each colony were analyzed by restriction digestion. The cloning efficiencies were calculated as the ratio of correct colonies to the total number of checked colonies. Each experiment was performed in three biological replicates and data are presented as mean values ± standard error (SEM). c Summary of results for cloning uncharacterized BGCs using CAPTURE. BGCs ranging from 10 to 113 kb can be robustly cloned using the CAPTURE method at close to 100% efficiency regardless of their GC-content. Source data are provided as a Source Data file.

    Techniques Used: Hybridization, Clone Assay, Purification

    Development of the CAPTURE method. a Overview of the workflow. In the first step, purified genomic DNA is digested by Cas12a enzyme to release the target BGC fragment. In the second step, digestion products are mixed with two DNA receivers containing lox P sites at their ends. The target BGC fragment and DNA receivers are assembled together using T4 DNA polymerase exo + fill-in DNA assembly. In the final step, the assembly mixture is transformed into E. coli cells harboring a circularization helper plasmid. The linear DNA is able to circularize in vivo by Cre- lox recombination. b DNA map of helper plasmid pBE14. tcr : tetracycline resistance marker; araBAD : L-arabinose inducible promoter and its regulator; gam : phage lambda Red gam gene; pSC101: temperature-sensitive origin of replication; recA1 : mutated E. coli recA gene to increase transformation efficiency. c Comparison of recombination frequency between Flp (pBE11) and Cre (pBE12) helper plasmids. -: without L-arabinose induction, +: with L-arabinose induction. Recombination frequencies were calculated based on the ratio of white colonies to the total number of acquired colonies. d Linear DNA transformation efficiency for E. coli cells harboring pBE11 (Flp), pBE12 (Cre), pBE14 (Cre and recA1) helper plasmids. Both pBE12 and pBE14 E. coli cells exhibited transformation efficiencies similar to circular DNA. e Comparison of in vitro versus in vivo circularization for two large (50 kb, 73 kb) linear DNA molecules. In vivo circularization showed ~33-fold and 150-fold higher frequency than in vitro circularization for 50 kb and 73 kb molecules, respectively. Circularization frequencies were calculated based on the number of colonies acquired for each circularization experiment in comparison to the number of colonies acquired after transformation of the original circular DNA (see Methods for full description). Each experiment was performed in three biological replicates and data are presented as mean values ± standard deviation (SD). Source data are provided as a Source Data file.
    Figure Legend Snippet: Development of the CAPTURE method. a Overview of the workflow. In the first step, purified genomic DNA is digested by Cas12a enzyme to release the target BGC fragment. In the second step, digestion products are mixed with two DNA receivers containing lox P sites at their ends. The target BGC fragment and DNA receivers are assembled together using T4 DNA polymerase exo + fill-in DNA assembly. In the final step, the assembly mixture is transformed into E. coli cells harboring a circularization helper plasmid. The linear DNA is able to circularize in vivo by Cre- lox recombination. b DNA map of helper plasmid pBE14. tcr : tetracycline resistance marker; araBAD : L-arabinose inducible promoter and its regulator; gam : phage lambda Red gam gene; pSC101: temperature-sensitive origin of replication; recA1 : mutated E. coli recA gene to increase transformation efficiency. c Comparison of recombination frequency between Flp (pBE11) and Cre (pBE12) helper plasmids. -: without L-arabinose induction, +: with L-arabinose induction. Recombination frequencies were calculated based on the ratio of white colonies to the total number of acquired colonies. d Linear DNA transformation efficiency for E. coli cells harboring pBE11 (Flp), pBE12 (Cre), pBE14 (Cre and recA1) helper plasmids. Both pBE12 and pBE14 E. coli cells exhibited transformation efficiencies similar to circular DNA. e Comparison of in vitro versus in vivo circularization for two large (50 kb, 73 kb) linear DNA molecules. In vivo circularization showed ~33-fold and 150-fold higher frequency than in vitro circularization for 50 kb and 73 kb molecules, respectively. Circularization frequencies were calculated based on the number of colonies acquired for each circularization experiment in comparison to the number of colonies acquired after transformation of the original circular DNA (see Methods for full description). Each experiment was performed in three biological replicates and data are presented as mean values ± standard deviation (SD). Source data are provided as a Source Data file.

    Techniques Used: Purification, Transformation Assay, Plasmid Preparation, In Vivo, Marker, In Vitro, Standard Deviation

    8) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    9) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    10) Product Images from "Demonstration of a universal surface DNA computer"

    Article Title: Demonstration of a universal surface DNA computer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh635

    Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.
    Figure Legend Snippet: Computation of a simple circuit consisting of a NOR gate and an OR gate. ( a ) DNAs encoding the inputs were attached to the surfaces by the 3′ thiol group. ‘FFX’ designates the input DNAs in which X 1 is FALSE, X 2 is FALSE and X3 can be either TRUE or FALSE (i.e. input A and B). P indicates a phosphate group and S the underlying surface chemistry. ( b ) Complements to the words (X 1 is F) and (X 2 is F) were added to the surfaces and allowed to hybridize with the attached DNAs. ( c ) T4 DNA ligase linked the two complements hybridized with the ‘FFX’ DNAs. ( d ) Differential melting melted off hybridized one-word complements and left the linked two-word complements in place. ( e ) In the ‘FFX’ DNAs, polymerase extended the two-word complements to their 3′ ends. A TRUE fourth word (X 4 is T) was appended to the distal end of the now double-stranded ‘FFX’ DNAs. Note here that the 5′ end of the newly attached word was not phosphorylated, whereas the 5′ end of its complement was phosphorylated. This was designed so that only one copy of this word would be appended. ( f ) The complements to the words (X 1 is T) and (X 2 is T) were added to the surface and allowed to hybridize. ( g ) The complements added in (f) were extended by polymerase. Then a FALSE fourth word was appended to the surfaces by ligation. Note again that the design of this one-bit word was such that only the 5′ end of its complementary strand was phosphorylated for the same reason as for (e). The lack of a 5′ phosphate at the ends of the ‘FFX’ DNAs also ensured that this FALSE fourth word would not be appended to them. ( h ) DNAs after the NOR gate computation. Note here that the DNAs were now without a phosphate group. A kinase phosphorylated them before the OR gate computation. ( i ) DNAs in which (X 3 is F) and (X 4 is F) were MARKED in the first APPEND-MARKED operation for the OR gate. ( j ) A FALSE fifth word, rather than a TRUE fifth word, was appended. The change is equivalent to a NOT gate. ( k ) DNAs after the OR gate computation.

    Techniques Used: Ligation

    11) Product Images from "Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform"

    Article Title: Analysis of polyclonal vector integration sites using Nanopore sequencing as a scalable, cost-effective platform

    Journal: bioRxiv

    doi: 10.1101/833897

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are 4 restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3’LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5’ overhangs: 5’-CATG-3’, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3’LTR and the 3’LTR/distal transgene junction. This is followed by nested PCR, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3’ end and a 5’overhang (5’-ATG-3’). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3’ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3’LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a high annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3’LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

    12) Product Images from "Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues"

    Article Title: Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues

    Journal: Biochemistry

    doi: 10.1021/bi802269f

    In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical
    Figure Legend Snippet: In vitro assay of DNA flexibility enhancement by HMGB proteins and chimeras. A. Example data from T4 DNA ligase cyclization assay for 200-bp DNA probe in the absence (—) and presence of 40 nM HMGB constructs 16 and 5 (see ). B. Graphical

    Techniques Used: In Vitro, Construct

    13) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).
    Figure Legend Snippet: Average percentage of sequences carrying tag-jumps across amplicon pools built into Illumina libraries with four different library protocol treatments; +/+: T4 DNA polymerase blunt-ending and post-ligation PCR; −/+: no T4 DNA polymerase blunt-ending, with post-ligation PCR; +/−: T4 DNA polymerase blunt-ending and no post-ligation PCR; −/−: no T4 DNA polymerase blunt-ending and no post-ligation PCR (Tagsteady protocol) (n=6 for +/+, −/+, +/−, −/−). To mimic the effect of large amounts of single-stranded DNA generated in the metabarcoding PCR, aliquots of four of the amplicon pools were denatured and subsequently re-hybridized to form double-stranded DNA. These were then built into libraries with the −/− and +/− protocols (n=4 for d+/− and d−/−. Asterisks (*) denotes statistical significant difference between treatments (unpaired t-test, α=0.05).

    Techniques Used: Amplification, Ligation, Polymerase Chain Reaction, Generated

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.
    Figure Legend Snippet: Experimental overview. 1A) Six pools of 5’ twin-tagged amplicons generated with three metabarcoding primer sets were used to assess the effect of blunt-ending and post-ligation PCR on tag-jumps. Each of the six amplicon pools were subjected to four different treatments. 1B) The four treatments represent combinations with and without T4 DNA Polymerase blunt-ending in the end-repair step and 1C) with and without post-ligation PCR. 1D) This resulted in 24 libraries for the 6 amplicon pools, representing four library preparation treatments for each amplicon pool. 2A) To further assess the effect of T4 DNA Polymerase blunt-ending on the prevalence of tag-jumps, we denatured and re-hybridised four amplicon pools (16sMam1/2 and 16sIns1/2). 2B) End-repair was carried out with and without T4 DNA Polymerase blunt-ending and with no post-ligation PCR (2C). 3) Finally, to validate the robustness and stability of the Tagsteady protocol, we applied it to 15 pools of twin-tagged amplicons.

    Techniques Used: Generated, Ligation, Polymerase Chain Reaction, Amplification

    14) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    15) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    16) Product Images from "Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging"

    Article Title: Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac300

    Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.
    Figure Legend Snippet: Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.

    Techniques Used: Polymerase Chain Reaction, Synthesized, Incubation

    Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.
    Figure Legend Snippet: Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.

    Techniques Used: Construct, Polymerase Chain Reaction, Incubation, Agarose Gel Electrophoresis

    Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.
    Figure Legend Snippet: Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.

    Techniques Used: Polymerase Cycling Assembly, Generated, Sequencing, Amplification, Polymerase Chain Reaction, Construct, Incubation

    17) Product Images from "Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy"

    Article Title: Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2019-000299

    Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.
    Figure Legend Snippet: Schematic for PCR amplification of flanking genomic sequences. (A) Genomic DNA is digested with two 6-cutter restriction enzymes, NcoI and BspHI , which together are anticipated to cut at approximately 2 kb intervals. There are four restriction sites within the transgene sequence, the most distal of which is 1185 bp from the 3′LTR / genomic junction. NcoI and BspHI generate identical 4-nucleotide 5′ overhangs: 5′-CATG-3′, which can be circularized for inverse PCR or ligated to linker cassettes. (B) Inverse PCR begins with circularization with T4 DNA ligase, followed by PCR amplification of the unknown flanking genomic sequences using primers targeting the 3′LTR and the 3′LTR/distal transgene junction, indicated by continuous arrows. This is followed by nested PCR, indicated by dotted arrows, which incorporates tailing sequences for subsequent barcoding. The combined lengths of the dotted lines in the inner circle indicate the minimum theoretical length prior to the addition of tailing sequences and barcodes. (C) The ligation cassette comprises two partially complementary strands: a 27-nucleotide strand and a 14-nucleotide strand, the latter with a mismatched A at the 3′ end and a 5′overhang (5′-ATG-3′). Before cassette ligation, the genomic DNA fragments are filled with a single ddCTP to prevent elongation or ligation at the recessed 3′ end. Cassette ligation results in a nick on this strand, indicated by ‘X’. During the first cycle of PCR, fragments containing flanking genomic DNA are amplified by a primer spanning the transgene/3′LTR. The longer cassette strand does not prime because its complementary shorter strand has not ligated; whereas the shorter cassette strand does not prime because only 10 nucleotides are complementary to the longer cassette strand, resulting in a low annealing temperature. This cassette design limits the amplification of non-flanking genomic DNA and reduces PCR blocking by the shorter cassette strand. Subsequent cycles are primed by both the transgene/3′LTR primer and the longer cassette strand.

    Techniques Used: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Inverse PCR, Nested PCR, Ligation, Blocking Assay

    18) Product Images from "Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging"

    Article Title: Efficient golden gate assembly of DNA constructs for single molecule force spectroscopy and imaging

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac300

    Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.
    Figure Legend Snippet: Large DNA hairpin construction. ( A ) Design of DNA hairpin with 1.5 kb duplex arm and 2.1 kb spacer between the bead and surface. ( B ) Four PCR amplicons are synthesized together with two oligonucleotide parts and incubated with BsaI and T4 DNA ligase. ( C ) Gel analysis of final product. Bands from the starting material PCR amplicons, together with intermediate products and the final full-length product are indicated. ( D ) Schematic of DNA hairpin assembled in a magnetic tweezers experiment. At high forces the hairpin section is unzipped. ( E ) Example traces from three separate beads showing force–extension curves during increasing and decreasing force ramps. ( F ) Single molecule unwinding events after addition of the helicase PcrA at a constant force of 11 pN.

    Techniques Used: Polymerase Chain Reaction, Synthesized, Incubation

    Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.
    Figure Legend Snippet: Construction of DNA with a short synthetic hairpin. ( A ) Design of construct with labelled biotin and digoxigenin handles and 75 bp synthetic hairpin at centre. ( B ) The design is assembled by separately preparing the PCR parts and oligo parts as described in Figure 1 before a one-pot incubation with BsaI and T4 DNA ligase. ( C ) Agarose gel image of product showing band corresponding to full-length construct. ( D ) Schematic of magnetic tweezers experiment and extension-time trace at 14 pN for one example bead. The extension shows spontaneous fluctuations between two states characteristic of the unfolding and refolding of the DNA hairpin.

    Techniques Used: Construct, Polymerase Chain Reaction, Incubation, Agarose Gel Electrophoresis

    Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.
    Figure Legend Snippet: Workflow for generating long duplex DNA and hairpin structures via golden gate assembly. PCR amplicons are generated with primer overhangs that code for the BsaI recognition site and a unique four letter sequence generated as a 5′ overhang after BsaI digestion (each four base overhang containing sequence and its reverse complement is represented by a different colour). For attachment to surfaces or beads, labelled dUTPs are included in the amplification step. Separately, synthetic oligonucleotide parts are annealed together to form different structures such as duplexes, connectors and hairpin loops. These oligonucleotide parts have four base overhangs designed to base pair with specific PCR amplicons. To form the final construct design, specific subsets of the PCR parts and oligonucleotide parts are incubated together with BsaI and T4 DNA ligase in a one pot reaction.

    Techniques Used: Polymerase Cycling Assembly, Generated, Sequencing, Amplification, Polymerase Chain Reaction, Construct, Incubation

    19) Product Images from "Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling"

    Article Title: Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity and bias profiling

    Journal: bioRxiv

    doi: 10.1101/322297

    Assay results for the ligation of randomized four-base overhangs by T4 DNA Ligase. SMRT sequencing results for ligating 100 nM of the multiplexed four-base overhang substrate 18h at 25°C, with 1.75 μM T4 DNA ligase in standard ligation buffer. Observations have been normalized to 100,000 ligation events (see Supporting Data for actual observation totals). (A) Frequency heat map of all ligation events (log-scaled). Overhangs are listed alphabetically left to right (AAAA, AAAC…TTTG,, TTTT) and bottom to top such that the Watson-Crick pairings are shown on the diagonal. (B) Stacked bar plot showing the frequency of ligation products containing each overhang, corresponding to each row in the heat map in (A). Fully Watson-Crick paired ligation results are indicated in blue, and ligation products containing one or more mismatches are in orange.
    Figure Legend Snippet: Assay results for the ligation of randomized four-base overhangs by T4 DNA Ligase. SMRT sequencing results for ligating 100 nM of the multiplexed four-base overhang substrate 18h at 25°C, with 1.75 μM T4 DNA ligase in standard ligation buffer. Observations have been normalized to 100,000 ligation events (see Supporting Data for actual observation totals). (A) Frequency heat map of all ligation events (log-scaled). Overhangs are listed alphabetically left to right (AAAA, AAAC…TTTG,, TTTT) and bottom to top such that the Watson-Crick pairings are shown on the diagonal. (B) Stacked bar plot showing the frequency of ligation products containing each overhang, corresponding to each row in the heat map in (A). Fully Watson-Crick paired ligation results are indicated in blue, and ligation products containing one or more mismatches are in orange.

    Techniques Used: Ligation, Sequencing

    Frequency of specific base pair mismatches by position. Incidence of each possible mismatched base pair observed during ligation of four-base overhangs, with 100 nM of the multiplexed substrate, 1.75 μM T4 DNA ligase, and 18 h incubation at 25°C in standard ligation buffer. This figure was generated from the same data as shown in Figures 2 . (A) shows the results for the edge position (N1:N4’); (B) for the middle position (N2:N3′).
    Figure Legend Snippet: Frequency of specific base pair mismatches by position. Incidence of each possible mismatched base pair observed during ligation of four-base overhangs, with 100 nM of the multiplexed substrate, 1.75 μM T4 DNA ligase, and 18 h incubation at 25°C in standard ligation buffer. This figure was generated from the same data as shown in Figures 2 . (A) shows the results for the edge position (N1:N4’); (B) for the middle position (N2:N3′).

    Techniques Used: Ligation, Incubation, Generated

    Schematic of multiplexed ligation fidelity and bias profiling assay. (A) Libraries containing randomized four-base overhangs were synthesized and ligated with T4 DNA ligase under various conditions. The hairpin substrates contain the Pacific-Biosciences SMRTbell adapter sequence, an internal 6-base random barcode used to confirm strand identity and monitor the substrate sequence bias derived from oligonucleotide synthesis, and randomized four-base overhangs. (B) Ligated substrates form circular molecules, in which a double-stranded insert DNA is capped with SMRTbell adapters. These products were sequenced utilizing Pacific Biosciences SMRT sequencing, which produced long rolling-circle sequencing reads. Consensus sequences were built for the top and bottom strands independently, allowing extraction of the overhang identity and barcode sequence.
    Figure Legend Snippet: Schematic of multiplexed ligation fidelity and bias profiling assay. (A) Libraries containing randomized four-base overhangs were synthesized and ligated with T4 DNA ligase under various conditions. The hairpin substrates contain the Pacific-Biosciences SMRTbell adapter sequence, an internal 6-base random barcode used to confirm strand identity and monitor the substrate sequence bias derived from oligonucleotide synthesis, and randomized four-base overhangs. (B) Ligated substrates form circular molecules, in which a double-stranded insert DNA is capped with SMRTbell adapters. These products were sequenced utilizing Pacific Biosciences SMRT sequencing, which produced long rolling-circle sequencing reads. Consensus sequences were built for the top and bottom strands independently, allowing extraction of the overhang identity and barcode sequence.

    Techniques Used: Ligation, Synthesized, Sequencing, Derivative Assay, Oligonucleotide Synthesis, Produced

    Predicted versus observed fragment linkages in Golden-Gate assembly of the HF and LF 10-fragment assemblies. Junction overhangs can be found in Table 1 . The intensity of the color corresponds to the number of instances of that junction observed in a Pacific Biosciences SMRT sequencing experiment, normalized to 100,000 total junctions. Predicted frequencies of junctions are based on the fidelity library data generated for the four-base overhang substrate ligated with T4 DNA ligase at 25°C for 18 h. The experimental observations shown are for assembly of the 10-fragment HF and LF sets with Golden Gate Assembly mix, 37°C 5 min/16°C 5 min, 30 cycles.
    Figure Legend Snippet: Predicted versus observed fragment linkages in Golden-Gate assembly of the HF and LF 10-fragment assemblies. Junction overhangs can be found in Table 1 . The intensity of the color corresponds to the number of instances of that junction observed in a Pacific Biosciences SMRT sequencing experiment, normalized to 100,000 total junctions. Predicted frequencies of junctions are based on the fidelity library data generated for the four-base overhang substrate ligated with T4 DNA ligase at 25°C for 18 h. The experimental observations shown are for assembly of the 10-fragment HF and LF sets with Golden Gate Assembly mix, 37°C 5 min/16°C 5 min, 30 cycles.

    Techniques Used: Sequencing, Generated

    20) Product Images from "Methylase-assisted subcloning for high throughput BioBrick assembly"

    Article Title: Methylase-assisted subcloning for high throughput BioBrick assembly

    Journal: PeerJ

    doi: 10.7717/peerj.9841

    4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .
    Figure Legend Snippet: 4R/2M (PstI) BioBrick assembly. The EcoRI site of the recipient plasmid (1) and SpeI site of the insert (2) are methylated in vivo. The recipient plasmid (1) is digested with SpeI and PstI, so that it releases a short 18 bp stuffer (or “snippet”, 4). The donor plasmid (2) is separately digested with XbaI and PstI, producing the desired insert (5) and the undesired donor plasmid fragment (6). The restriction enzymes are heat-killed, the digestion products are mixed and reacted with T4 DNA ligase, forming three sets of ligation products: parental-plasmids (1-2), homodimers (7-9) and heterodimers (10-13). The 36 bp snippet homodimer is not shown, nor are trimer, tetramer and other higher order products. The homodimer products (7-9) are large perfect inverted repeats, which are not expected to replicate efficiently in vivo. Moreover, none of the undesired parental (1-2), homodimer (7-9) or heterodimers (10-11) are resistant to subsequent digestion with EcoRI and SpeI. Only the desired insert/recipient recombinant plasmid (13) retains its ability to transform E. coli .

    Techniques Used: Plasmid Preparation, Methylation, In Vivo, Ligation, Recombinant

    2RM BioBrick assembly. The XbaI site of one cloned BioBrick part (1), and the SpeI site of another (2), are methylated in vivo. These methylated plasmids are mixed together with XbaI, SpeI and T4 DNA ligase. Each plasmid is digested by one restriction endonuclease and protected from the other. The linear digestion products (3-4) are self-ligated to form the parental plasmids (1-2), to another copy of the same molecule in one of two orientations to form a homodimer product (5-8) or to a copy of the other plasmid (again in one of two possible orientations) to form a heterodimer (9-10). The parental plasmids (1-2) and homodimers (5-8) are susceptible to re-digestion, so they are depleted over time while the heterodimers (9-10) accumulate. Both the desired (10) and undesired (9) heterodimers replicate in vivo so restriction mapping is required to differentiate between the two.
    Figure Legend Snippet: 2RM BioBrick assembly. The XbaI site of one cloned BioBrick part (1), and the SpeI site of another (2), are methylated in vivo. These methylated plasmids are mixed together with XbaI, SpeI and T4 DNA ligase. Each plasmid is digested by one restriction endonuclease and protected from the other. The linear digestion products (3-4) are self-ligated to form the parental plasmids (1-2), to another copy of the same molecule in one of two orientations to form a homodimer product (5-8) or to a copy of the other plasmid (again in one of two possible orientations) to form a heterodimer (9-10). The parental plasmids (1-2) and homodimers (5-8) are susceptible to re-digestion, so they are depleted over time while the heterodimers (9-10) accumulate. Both the desired (10) and undesired (9) heterodimers replicate in vivo so restriction mapping is required to differentiate between the two.

    Techniques Used: Clone Assay, Methylation, In Vivo, Plasmid Preparation

    21) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    22) Product Images from "Nucleic acid evolution and minimization by nonhomologous random recombination"

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    Journal: Nature biotechnology

    doi: 10.1038/nbt736

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Figure Legend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Techniques Used: Ligation, Binding Assay

    23) Product Images from "Spatial chromatin accessibility sequencing resolves next-generation genome architecture"

    Article Title: Spatial chromatin accessibility sequencing resolves next-generation genome architecture

    Journal: bioRxiv

    doi: 10.1101/2022.04.21.489011

    Selecting the compatible restriction enzymes. In the SCA-seq, we used the methyltransferase (EcoGII or M.CviPI) to label the open chromatin. Then we used the restriction enzyme to digest genome and prepare for the next step ligation. The EcoGII methyltransferase transferred the methyl group to the 6’ carbon in adenosine (m6A modification). Due to the high density of the adenosines on genome (∼25%), the abundant artificial m6A may impair the restriction enzymes. We used the EcoGII to treat the HEK293T genomic DNAs (m6A +), and leave the control (m6a -) untreated. We tested HindIII (a) PstI and NdeI (b) on the methylated and unmethylated genomic DNAs, and all these restriction enzymes were inhibited by the abundant m6A modifications. DpnI is the m6A dependent restriction enzymes, and only digested the genomic locus with m6A modification (a DpnI). NA was the genomic DNA control. By the similar process, we tested the HindIII (c for lambda DNA), DpnII and NIaIII (d for HEK293T genomic DNAs) on the GpC methylated DNAs, which were treated by methyltransferase M.CviPI. DpnII and NIaIII were not significantly inhibited by GpC methylation. In the next step, we examined if the digested products could be ligated by T4 DNA ligase (e).
    Figure Legend Snippet: Selecting the compatible restriction enzymes. In the SCA-seq, we used the methyltransferase (EcoGII or M.CviPI) to label the open chromatin. Then we used the restriction enzyme to digest genome and prepare for the next step ligation. The EcoGII methyltransferase transferred the methyl group to the 6’ carbon in adenosine (m6A modification). Due to the high density of the adenosines on genome (∼25%), the abundant artificial m6A may impair the restriction enzymes. We used the EcoGII to treat the HEK293T genomic DNAs (m6A +), and leave the control (m6a -) untreated. We tested HindIII (a) PstI and NdeI (b) on the methylated and unmethylated genomic DNAs, and all these restriction enzymes were inhibited by the abundant m6A modifications. DpnI is the m6A dependent restriction enzymes, and only digested the genomic locus with m6A modification (a DpnI). NA was the genomic DNA control. By the similar process, we tested the HindIII (c for lambda DNA), DpnII and NIaIII (d for HEK293T genomic DNAs) on the GpC methylated DNAs, which were treated by methyltransferase M.CviPI. DpnII and NIaIII were not significantly inhibited by GpC methylation. In the next step, we examined if the digested products could be ligated by T4 DNA ligase (e).

    Techniques Used: Ligation, Modification, Methylation, Lambda DNA Preparation, Gel Permeation Chromatography

    24) Product Images from "Mismatch discrimination and sequence bias during end-joining by DNA ligases"

    Article Title: Mismatch discrimination and sequence bias during end-joining by DNA ligases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac241

    Effect of PEG on ligation fidelity and bias for T4 DNA ligase, T7 DNA Ligase, and hLig3. The normalized ligation frequency and individual overhang fidelity was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase or hLig3 incubated 1 h at 25°C in NEBNext ® Quick Ligation Buffer ( Supplementary Figures S2F–H and File S1 for raw data).The ligation frequency and ligation fidelity of overhangs are grouped by GC content. For comparison to data in standard ligation buffer, the median value is indicated by a horizontal line (red for reactions in standard ligation buffer; black for reactions in PEG-containing buffer).
    Figure Legend Snippet: Effect of PEG on ligation fidelity and bias for T4 DNA ligase, T7 DNA Ligase, and hLig3. The normalized ligation frequency and individual overhang fidelity was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase or hLig3 incubated 1 h at 25°C in NEBNext ® Quick Ligation Buffer ( Supplementary Figures S2F–H and File S1 for raw data).The ligation frequency and ligation fidelity of overhangs are grouped by GC content. For comparison to data in standard ligation buffer, the median value is indicated by a horizontal line (red for reactions in standard ligation buffer; black for reactions in PEG-containing buffer).

    Techniques Used: Ligation, Generated, Sequencing, Incubation

    Ligation bias for T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase and PBCV-1 DNA ligase. The normalized ligation frequency of each overhang was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1h at 25°C in standard ligation buffer ( Supplementary Figures S2A-S2E and File S1 for raw data).T7 DNA ligase exhibits a significant ligation bias with few overhangs ligated very efficiently, and the majority of overhangs ligated with much lower efficiency. T4 DNA ligase and hLig3 exhibit more uniform ligation bias compared to T7 DNA ligase. GC-rich overhangs tend to ligate more efficiently compared to AT-rich overhangs. Each overhang is colored according to its GC content (0% – dark red, 25% – light red, 50% – gray, 75% – light blue, 100% – dark blue.
    Figure Legend Snippet: Ligation bias for T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase and PBCV-1 DNA ligase. The normalized ligation frequency of each overhang was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1h at 25°C in standard ligation buffer ( Supplementary Figures S2A-S2E and File S1 for raw data).T7 DNA ligase exhibits a significant ligation bias with few overhangs ligated very efficiently, and the majority of overhangs ligated with much lower efficiency. T4 DNA ligase and hLig3 exhibit more uniform ligation bias compared to T7 DNA ligase. GC-rich overhangs tend to ligate more efficiently compared to AT-rich overhangs. Each overhang is colored according to its GC content (0% – dark red, 25% – light red, 50% – gray, 75% – light blue, 100% – dark blue.

    Techniques Used: Ligation, Generated, Sequencing, Incubation

    Ligation fidelity for T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase and PBCV-1 DNA ligase. The fidelity for each of the 256 four-base overhangs was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1h at 25°C in standard ligation buffer ( Supplementary Figures S2A-S2E and File S1 for raw data). Ligation fidelity is defined as the percentage of correct (Watson–Crick) versus incorrect (mismatch) ligation events. A horizontal line indicates the median ligation fidelity of all overhangs for a particular ligase. Each overhang is colored according to its GC content (0% – dark red, 25% – light red, 50% – gray, 75% – light blue, 100% – dark blue).
    Figure Legend Snippet: Ligation fidelity for T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase and PBCV-1 DNA ligase. The fidelity for each of the 256 four-base overhangs was generated by SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1h at 25°C in standard ligation buffer ( Supplementary Figures S2A-S2E and File S1 for raw data). Ligation fidelity is defined as the percentage of correct (Watson–Crick) versus incorrect (mismatch) ligation events. A horizontal line indicates the median ligation fidelity of all overhangs for a particular ligase. Each overhang is colored according to its GC content (0% – dark red, 25% – light red, 50% – gray, 75% – light blue, 100% – dark blue).

    Techniques Used: Ligation, Generated, Sequencing, Incubation

    Positional mismatch profiles. The frequency of specific base pair mismatches by position was observed for ligation of four-base overhangs. The results shown are for SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1 h at 25°C in standard ligation buffer ( Supplementary Figures S2A–E and File S1 for raw data). (A, C, E, G and I) show the results for the edge position (N1:N4); (B, D, F, H, and J) show the results for the middle position (N2:N3). The overhang positions (N 1 , N 2 , N 3 , N 4 ) are numbered from 5′- to 3′- for each strand. Each position in N 1 :N 4 and N 2 :N 3 refers to bases in opposite strands. Note that as strand designation is arbitrary, all ligation products were counted in both orientations (top-to-bottom and bottom-to-top strand).
    Figure Legend Snippet: Positional mismatch profiles. The frequency of specific base pair mismatches by position was observed for ligation of four-base overhangs. The results shown are for SMRT sequencing of ligation reactions with 100 nM of the multiplexed four-base overhang substrate and 1.75 μM T4 DNA ligase, T7 DNA ligase, hLig3, T3 DNA ligase, or PBCV-1 DNA ligase incubated 1 h at 25°C in standard ligation buffer ( Supplementary Figures S2A–E and File S1 for raw data). (A, C, E, G and I) show the results for the edge position (N1:N4); (B, D, F, H, and J) show the results for the middle position (N2:N3). The overhang positions (N 1 , N 2 , N 3 , N 4 ) are numbered from 5′- to 3′- for each strand. Each position in N 1 :N 4 and N 2 :N 3 refers to bases in opposite strands. Note that as strand designation is arbitrary, all ligation products were counted in both orientations (top-to-bottom and bottom-to-top strand).

    Techniques Used: Ligation, Sequencing, Incubation

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    New England Biolabs t4 dna ligase reaction buffer
    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with <t>T4</t> DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna ligase buffer
    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new <t>T4</t> DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.
    T4 Dna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Journal: Cell

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    doi: 10.1016/j.cell.2017.11.008

    Figure Lengend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Article Snippet: YY1: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of YY1.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Journal: Langmuir : the ACS journal of surfaces and colloids

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    doi: 10.1021/acs.langmuir.8b01812

    Figure Lengend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Article Snippet: PAGE-purified oligos were purchased from IDT. ssDNA circles were prepared by annealing 5 μ M phosphorylated template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG) and 4.5 μ M biotinylated primer oligo (5/Biosg/TC TCC TCC TTC T) in 1× T4 ligase reaction buffer (NEB B0202S)., Oligos were heated to 75 °C for 5 min and cooled to 4 °C at a rate of −1 °C min−1 .

    Techniques: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Functional studies of genetic variants associated with human diseases in Notch signaling-related genes using Drosophila

    doi: 10.1007/978-1-0716-2201-8_19

    Figure Lengend Snippet: Generation of an amx-yellow[wing2+] homology directed repair donor construct using the Golden Gate cloning strategy. (A) In order to clone the upstream (UHA) and downstream homology arms (DHA) of the homology directed repair (HDR) donor construct, perform PCR using specific primers and genomic fly DNA. In addition to the segments that anneal with the genomic DNA, the primers designed here have features that facilitate the subcloning of these fragments using the Golden Gate strategy. (B) In addition to the two homology arms generated by PCR, this protocol requires two plasmids, one that provides the vector backbone of the final product (pBH vector shown on the left) and another that provides the yellow[wing2+] cassette. (C) Assembly of the amx-yellow[wing2+] HDR plasmid through the Golden Gate reaction. By mixing the UHA and DHA from (A) , the two plasmids from (B) , a type IIs restriction enzyme BsaI and a DNA ligase, the four segments will be assembled into one plasmid through repetitive digestion and ligation reactions based on the specific overhangs created by the BsaI digestion (shown as overhangs ① to ④).

    Article Snippet: 1 ul 10X T4 ligation buffer (NEB, B0202S).

    Techniques: Construct, Clone Assay, Polymerase Chain Reaction, Subcloning, Generated, Plasmid Preparation, Ligation

    Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Journal: Nucleic Acids Research

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    doi: 10.1093/nar/gkx533

    Figure Lengend Snippet: Structure-seq2 leads to a lower ligation bias. ( A ) After RT (Figure 1 , step 1A/1B), excess of the 27 nt primer (blue, top, right) is still present in the solution. During ligation (Figure 1 , step 3A/3B), this primer can also ligate to the 40 nt hairpin adaptor (pink) to form an unwanted 67 nt by-product which has no insert and so results in sequencing reads with no utility. ( B ) The complement of the first nucleotide after the adaptor sequence read during sequencing is the nucleotide that ligated to the adaptor. Our new T4 DNA ligase-based method (green, –DMS and pink, +DMS) substantially decreases ligation bias as compared to the previous Circligase-based method (blue). Percentages equaling the transcriptomic distribution of the four nucleotides (black) are ideal.

    Article Snippet: After renaturing the purified cDNA with betaine, polyethylene glycol 8000 (PEG 8000) and hairpin donor (5′-pTGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG-3′-Spacer, where ‘5′-p’ is a 5′ phosphate and ‘3′-Spacer’ is a 3-carbon linker), 10× T4 DNA ligase buffer and T4 DNA ligase (NEB) were added to give a final 10 μl reaction mixture containing 500 mM Betaine, 20% PEG 8000, 10 μM hairpin donor, 1× T4 DNA ligase buffer, and 400 U T4 DNA ligase.

    Techniques: Ligation, Sequencing

    Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Journal: Nucleic Acids Research

    Article Title: Structure-seq2: sensitive and accurate genome-wide profiling of RNA structure in vivo

    doi: 10.1093/nar/gkx533

    Figure Lengend Snippet: Two versions of Structure-seq2 produce high quality data. In Structure-seq2, RNA (kelly green) is first modified by DMS or another chemical that can be read-out through reverse transcription. The RNA is then prepared for Illumina NGS sequencing by conversion to cDNA (Step 1A/1B, blue), ligating an adaptor (Step 3A/3B), and amplifying the products while incorporating TruSeq primer sequences (Step 5A/5B). In order to increase library quality, numerous improvements were made to the original Structure-seq protocol (boxed). These include performing the ligation with a hairpin adaptor and T4 DNA ligase (Step 3A/3B; pink) ( 10 ), and adding various purification steps to remove a deleterious by-product (Figure 2A ). We present two options for purification: PAGE purification ( A ) or a biotin–streptavidin pull down ( B ). In the PAGE purification method, an additional PAGE purification step is added after reverse transcription (Step 2A). In the biotin–streptavidin pull down method, biotinylated dNTPs (cyan) are incorporated into the extended product during reverse transcription (Step 1B) and are purified via a magnetic streptavidin pull down after reverse transcription (Step 2B) and after ligation (Step 4B). There is also a common, final PAGE purification step following amplification (Step 5A/5B). Finally, a custom sequencing primer (light green) is used during sequencing (Step 7A/7B) to further provide high quality data. Supplementary Figure S1 is a version of this figure with all the nucleotides shown explicitly.

    Article Snippet: After renaturing the purified cDNA with betaine, polyethylene glycol 8000 (PEG 8000) and hairpin donor (5′-pTGAAGAGCCTAGTCGCTGTTCANNNNNNCTGCCCATAGAG-3′-Spacer, where ‘5′-p’ is a 5′ phosphate and ‘3′-Spacer’ is a 3-carbon linker), 10× T4 DNA ligase buffer and T4 DNA ligase (NEB) were added to give a final 10 μl reaction mixture containing 500 mM Betaine, 20% PEG 8000, 10 μM hairpin donor, 1× T4 DNA ligase buffer, and 400 U T4 DNA ligase.

    Techniques: Modification, Next-Generation Sequencing, Sequencing, Ligation, Purification, Polyacrylamide Gel Electrophoresis, Amplification