large klenow fragment  (New England Biolabs)


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    Klenow Fragment 3 5 exo
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    New England Biolabs large klenow fragment
    Klenow Fragment 3 5 exo
    Klenow Fragment 3 5 exo 1 000 units
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    1) Product Images from "Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis"

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    Journal: bioRxiv

    doi: 10.1101/790014

    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.
    Figure Legend Snippet: Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Techniques Used: Selection, Marker, In Vitro, Transformation Assay, Sequencing, Ligation

    Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.
    Figure Legend Snippet: Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Techniques Used: Generated, In Vitro, Sequencing, Variant Assay, Ligation

    2) Product Images from "Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma"

    Article Title: Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

    Journal: Human Genomics

    doi: 10.1186/s40246-018-0160-8

    A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]
    Figure Legend Snippet: A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Techniques Used: Ligation

    3) Product Images from "Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment"

    Article Title: Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.67.12.5780-5790.2001

    Quantitative analysis of functional gene arrays. (A) Relationship of hybridization signal intensity to DNA target concentration from a single pure culture. Genomic DNA from nirS -containing P. stutzeri E4-2 was labeled with Cy5 and hybridized to the microarrays at the following target concentrations: 0.5, 1, 2.5, 5, 10, 25, 50, and 100 ng. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration. (B) Relationship of hybridization signal intensity to DNA target concentration using a mixture of target DNAs. The PCR products from the following nine strains were mixed together in different quantities (in picograms): E4-2 ( nirS ), 1,000; G179 ( nirK ), 500; wc301–37 ( amoA ), 250; ps-47 ( amoA ), 125; pB49 ( nirS ), 62.5; Y32K ( nirK ), 31.3; wA15 ( nirS ), 15.6; ps-80 ( amoA ), 7.8; wB54 ( nirK ), 3.9. All of these genes are less than 80% identical. The mixed templates were labeled with Cy5. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration for each strain. (C) Effects of probe DNA size and composition on hybridization signal intensity. Microarrays contained DNA fragments (200 ng μl −1 ) of different sizes amplified from different regions in the S. oneidensis MR-1 genome. The target DNA was prepared by labeling MR-1 genomic DNA with Cy5 using the Klenow fragment with random hexamer primers. For panels A and B, the data points are mean values derived from three independent microarray slides, with three replicates on each slide (a total of nine data points). Error bars showing the standard deviations are presented.
    Figure Legend Snippet: Quantitative analysis of functional gene arrays. (A) Relationship of hybridization signal intensity to DNA target concentration from a single pure culture. Genomic DNA from nirS -containing P. stutzeri E4-2 was labeled with Cy5 and hybridized to the microarrays at the following target concentrations: 0.5, 1, 2.5, 5, 10, 25, 50, and 100 ng. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration. (B) Relationship of hybridization signal intensity to DNA target concentration using a mixture of target DNAs. The PCR products from the following nine strains were mixed together in different quantities (in picograms): E4-2 ( nirS ), 1,000; G179 ( nirK ), 500; wc301–37 ( amoA ), 250; ps-47 ( amoA ), 125; pB49 ( nirS ), 62.5; Y32K ( nirK ), 31.3; wA15 ( nirS ), 15.6; ps-80 ( amoA ), 7.8; wB54 ( nirK ), 3.9. All of these genes are less than 80% identical. The mixed templates were labeled with Cy5. The plot shows the log-transformed average hybridization intensity versus the log-transformed target DNA concentration for each strain. (C) Effects of probe DNA size and composition on hybridization signal intensity. Microarrays contained DNA fragments (200 ng μl −1 ) of different sizes amplified from different regions in the S. oneidensis MR-1 genome. The target DNA was prepared by labeling MR-1 genomic DNA with Cy5 using the Klenow fragment with random hexamer primers. For panels A and B, the data points are mean values derived from three independent microarray slides, with three replicates on each slide (a total of nine data points). Error bars showing the standard deviations are presented.

    Techniques Used: Functional Assay, Hybridization, Concentration Assay, Labeling, Transformation Assay, Polymerase Chain Reaction, Amplification, Random Hexamer Labeling, Derivative Assay, Microarray

    The normalized distribution of signal intensity levels for nirS, nirK , and amoA genes as determined by microarray-based analysis of community DNA from marine sediment (W303) and surface soil (O22) environments. Bulk community DNA isolated from two different environmental samples was directly labeled with Cy5 using Klenow fragment with random hexamer primers and hybridized with the microarrays at 65°C in separate experiments. The hybridization signal intensity for each gene is presented. Shaded bars represent probes from environmental clones, and striped boxes represent pmo A probes from environmental clones. Open bars designate probes from pure cultures. The data represent mean values obtained from nine replicates after subtracting background hybridization to yeast genes. For the nirS graphs, bars 1 to 42 correspond to individual genes S1-S42 (see assigned designation in Table 1 on the web site). Similarly, bars 1 to 18 for nirK correspond to genes K1 to K18, and bars 1 to 22 for amoA and pmoA represent genes M1 to M22 (see our table on the website cited in Materials and Methods). The standard deviation of signal intensity is indicated on the top of each bar.
    Figure Legend Snippet: The normalized distribution of signal intensity levels for nirS, nirK , and amoA genes as determined by microarray-based analysis of community DNA from marine sediment (W303) and surface soil (O22) environments. Bulk community DNA isolated from two different environmental samples was directly labeled with Cy5 using Klenow fragment with random hexamer primers and hybridized with the microarrays at 65°C in separate experiments. The hybridization signal intensity for each gene is presented. Shaded bars represent probes from environmental clones, and striped boxes represent pmo A probes from environmental clones. Open bars designate probes from pure cultures. The data represent mean values obtained from nine replicates after subtracting background hybridization to yeast genes. For the nirS graphs, bars 1 to 42 correspond to individual genes S1-S42 (see assigned designation in Table 1 on the web site). Similarly, bars 1 to 18 for nirK correspond to genes K1 to K18, and bars 1 to 22 for amoA and pmoA represent genes M1 to M22 (see our table on the website cited in Materials and Methods). The standard deviation of signal intensity is indicated on the top of each bar.

    Techniques Used: Microarray, Isolation, Environmental Sampling, Labeling, Random Hexamer Labeling, Hybridization, Clone Assay, Standard Deviation

    4) Product Images from "Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis"

    Article Title: Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17061-3

    Schematic outline of TRIAD. a Generation of deletion libraries. Step 1: The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence on circular plasmid DNA. Step 2: MlyI digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a: self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 14 . Step 3b: DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b: MlyI digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b: self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. b Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by NotI and MlyI removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins3 and Ins3 (with, respectively, 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3′-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.
    Figure Legend Snippet: Schematic outline of TRIAD. a Generation of deletion libraries. Step 1: The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence on circular plasmid DNA. Step 2: MlyI digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a: self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 14 . Step 3b: DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b: MlyI digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b: self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. b Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by NotI and MlyI removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins3 and Ins3 (with, respectively, 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3′-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Techniques Used: Generated, In Vitro, Sequencing, Plasmid Preparation, Variant Assay, Ligation

    Mechanism for the generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1 bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5′, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry, respectively, 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1, 2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5′CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3′, 2-base overhangs with the target DNA (i.e., 5′N 5 T on one end and 5′TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.
    Figure Legend Snippet: Mechanism for the generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1 bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5′, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry, respectively, 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1, 2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5′CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3′, 2-base overhangs with the target DNA (i.e., 5′N 5 T on one end and 5′TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Techniques Used: Sequencing, Ligation

    5) Product Images from "Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells"

    Article Title: Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-015-0205-1

    Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products
    Figure Legend Snippet: Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products

    Techniques Used: Flow Cytometry, Modification, Produced, Amplification, Polymerase Chain Reaction

    6) Product Images from "Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction"

    Article Title: Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2016.05.017

    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).
    Figure Legend Snippet: Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).

    Techniques Used: Concentration Assay, Activity Assay, Sequencing, Binding Assay

    Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).
    Figure Legend Snippet: Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).

    Techniques Used: DNA Synthesis

    Related Articles

    Concentration Assay:

    Article Title: An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli
    Article Snippet: .. To facilitate the re-ligation (see below), the purified DNA was blunt-ended with 3.2 U Klenow fragment in 10× NEBuffer 2 (New England Biolabs) and dNTPs (final concentration 33 μM each nucleotide) for 15 min at 25°C. .. Then EDTA was added to a final concentration of 10 mM, and the tube was incubated at 75°C for 20 min (Figure ).

    Purification:

    Article Title: An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli
    Article Snippet: .. To facilitate the re-ligation (see below), the purified DNA was blunt-ended with 3.2 U Klenow fragment in 10× NEBuffer 2 (New England Biolabs) and dNTPs (final concentration 33 μM each nucleotide) for 15 min at 25°C. .. Then EDTA was added to a final concentration of 10 mM, and the tube was incubated at 75°C for 20 min (Figure ).

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

    other:

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase
    Article Snippet: Both polymerases could replace UL30-UL42, with Klenow Fragment generating products ~1 kB long.

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase
    Article Snippet: The absence of any products longer than ~100 nucleotides in assays containing UL30 is not due to either the lower processivity of UL30 relative to UL30-UL42 since UL30 and Klenow Fragment have similar processivity ( ) and Klenow Fragment generates long products.

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase
    Article Snippet: Even without ICP8, we found that non-cognate polymerases such as Klenow Fragment and T4 DNA polymerase allow the herpes helicase to more efficiently unwind DNA and allow the synthesis of long products.

    DNA Synthesis:

    Article Title: 5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs
    Article Snippet: .. However, contrary to the gp43-gp41 replication couple, DNA synthesis performed by Klenow fragment was not stimulated by gp41 ( , compare lanes 2, 3, 5, and 6). .. Under both conditions (with or without gp41), DNA synthesis was highly distributive as indicated by the numerous pause sites that were clearly visible along the template strand.

    Ligation:

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions
    Article Snippet: .. To allow for ligation with the blunt-end of the double-stranded adaptors, 15 µl of purified MNase digested DNA fragments were blunt-ended in a final volume of 20 µl by Klenow fragment of Escherichia coli DNA polymerase (New England Biolabs, CA, USA) and phosphorylated by T4 polynucleotide kinase (New England Biolabs) in a final volume of 30 µl. .. Adaptor ligation The blunt-ended and phosphorylated DNA fragments were ligated to the double-stranded adaptors A and B as described in ( ) with the following modifications.

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  • 99
    New England Biolabs klenow large fragment
    Primer extension using AHP dUTP (1.5 h). ( A ) Template T2 and primer P3. ( B ) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), <t>Klenow</t> (Kl, 37°C), KOD (KO, 72°C) and <t>Therminator™</t> II (Th, 72°C) polymerases. Lane P: primer P3. ( C ) Reactions using Gotaq polymerase at 60°C. ( D ) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).
    Klenow Large Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t7 endonuclease i
    T7 endonuclease I assay shows similar efficiencies of HVEM gene CRISPR-Cas9 modification by different sgRNAs (1, 3, 6 and 13) evaluated. (A) Undigested HVEM band amplified by PCR (cleavage negative control). (B) The resulting digestions of T7/EI assay (red arrows) revealed a similar cleavage pattern to each sgRNA because they presented percentages of HVEM gene modification very similar.
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 endonuclease i/product/New England Biolabs
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    Primer extension using AHP dUTP (1.5 h). ( A ) Template T2 and primer P3. ( B ) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), Klenow (Kl, 37°C), KOD (KO, 72°C) and Therminator™ II (Th, 72°C) polymerases. Lane P: primer P3. ( C ) Reactions using Gotaq polymerase at 60°C. ( D ) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).

    Journal: Nucleic Acids Research

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    doi: 10.1093/nar/gkw028

    Figure Lengend Snippet: Primer extension using AHP dUTP (1.5 h). ( A ) Template T2 and primer P3. ( B ) Twenty percent denaturing PAGE analysis of reactions using Gotaq (Go, 72°C), Klenow (Kl, 37°C), KOD (KO, 72°C) and Therminator™ II (Th, 72°C) polymerases. Lane P: primer P3. ( C ) Reactions using Gotaq polymerase at 60°C. ( D ) Mass spectrum of AHP-modified fully extended product using Klenow (calculated mass: 10621).

    Article Snippet: Klenow large fragment, Therminator™ II, M-MuLV (RNase H− ) reverse transcriptase, AMV reverse transcriptase, RNase inhibitor and λ-exonuclease were purchased from New England Biolabs.

    Techniques: Polyacrylamide Gel Electrophoresis, Modification

    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Selection, Marker, In Vitro, Transformation Assay, Sequencing, Ligation

    Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Generated, In Vitro, Sequencing, Variant Assay, Ligation

    A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Journal: Human Genomics

    Article Title: Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

    doi: 10.1186/s40246-018-0160-8

    Figure Lengend Snippet: A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Article Snippet: DNA Polymerase I Large (Klenow) Fragment, restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs (NEB), USA. dNTP mix was purchased from Promega, USA.

    Techniques: Ligation

    T7 endonuclease I assay shows similar efficiencies of HVEM gene CRISPR-Cas9 modification by different sgRNAs (1, 3, 6 and 13) evaluated. (A) Undigested HVEM band amplified by PCR (cleavage negative control). (B) The resulting digestions of T7/EI assay (red arrows) revealed a similar cleavage pattern to each sgRNA because they presented percentages of HVEM gene modification very similar.

    Journal: bioRxiv

    Article Title: The Role of Hvem and its Interaction with Btla and Cd160 in b-Cell Lymphoma Progression

    doi: 10.1101/754291

    Figure Lengend Snippet: T7 endonuclease I assay shows similar efficiencies of HVEM gene CRISPR-Cas9 modification by different sgRNAs (1, 3, 6 and 13) evaluated. (A) Undigested HVEM band amplified by PCR (cleavage negative control). (B) The resulting digestions of T7/EI assay (red arrows) revealed a similar cleavage pattern to each sgRNA because they presented percentages of HVEM gene modification very similar.

    Article Snippet: Annealed DNA products were digested by T7 endonuclease I (10 U) (NEB, M0302) (37°C for 30 min).

    Techniques: T7EI Assay, CRISPR, Modification, Amplification, Polymerase Chain Reaction, Negative Control