klenow fragment  (New England Biolabs)


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    Structured Review

    New England Biolabs klenow fragment
    Force dependence of the replication rate. ( a ) Replication by <t>Klenow</t> on a charomid is started at 3 pN. Increasing the force to 24 pN stops the replication. After reducing the force back to 3 pN, the polymerization degree, N , has not changed and the enzyme starts replicating again. ( b ) Number of bases N ( t ) replicated by Sequenase on pXΔII at two different forces: 1 pN and 16 pN. ( c ) Mean replication rate, 〈 v 〉, versus force for Sequenase: high stringency hybridization ( K = 13, J = 154; ○) and random priming ( K = 5, J = 82; ⋄). The error bars represent σ 〈 v 〉 estimated as explained in Materials and Methods . The full curve is a fit to the model described in the text 〈 v ( F )〉 = v 0 exp(− nF Δ h / k B T ), where v 0 = 200 b/s and n = 2.1 (only ○ were fitted). The dashed curve is obtained with the previously determined v 0 and n = 1 (as expected if only one base was rate determining). ( d ) Replication rate versus force for Klenow: low stringency hybridization ( K = 24, J = 298; ⋄), high stringency hybridization ( K = 9, J = 114; ○). Replication rate for Klenow minus <t>exo3′→5′</t> high stringency hybridization ( K = 4, J = 50; □). The full curve is a fit to ⋄, where v 0 = 13.5 b/s and n = 4.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Replication by a single DNA polymerase of a stretched single-stranded DNA"

    Article Title: Replication by a single DNA polymerase of a stretched single-stranded DNA

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Force dependence of the replication rate. ( a ) Replication by Klenow on a charomid is started at 3 pN. Increasing the force to 24 pN stops the replication. After reducing the force back to 3 pN, the polymerization degree, N , has not changed and the enzyme starts replicating again. ( b ) Number of bases N ( t ) replicated by Sequenase on pXΔII at two different forces: 1 pN and 16 pN. ( c ) Mean replication rate, 〈 v 〉, versus force for Sequenase: high stringency hybridization ( K = 13, J = 154; ○) and random priming ( K = 5, J = 82; ⋄). The error bars represent σ 〈 v 〉 estimated as explained in Materials and Methods . The full curve is a fit to the model described in the text 〈 v ( F )〉 = v 0 exp(− nF Δ h / k B T ), where v 0 = 200 b/s and n = 2.1 (only ○ were fitted). The dashed curve is obtained with the previously determined v 0 and n = 1 (as expected if only one base was rate determining). ( d ) Replication rate versus force for Klenow: low stringency hybridization ( K = 24, J = 298; ⋄), high stringency hybridization ( K = 9, J = 114; ○). Replication rate for Klenow minus exo3′→5′ high stringency hybridization ( K = 4, J = 50; □). The full curve is a fit to ⋄, where v 0 = 13.5 b/s and n = 4.
    Figure Legend Snippet: Force dependence of the replication rate. ( a ) Replication by Klenow on a charomid is started at 3 pN. Increasing the force to 24 pN stops the replication. After reducing the force back to 3 pN, the polymerization degree, N , has not changed and the enzyme starts replicating again. ( b ) Number of bases N ( t ) replicated by Sequenase on pXΔII at two different forces: 1 pN and 16 pN. ( c ) Mean replication rate, 〈 v 〉, versus force for Sequenase: high stringency hybridization ( K = 13, J = 154; ○) and random priming ( K = 5, J = 82; ⋄). The error bars represent σ 〈 v 〉 estimated as explained in Materials and Methods . The full curve is a fit to the model described in the text 〈 v ( F )〉 = v 0 exp(− nF Δ h / k B T ), where v 0 = 200 b/s and n = 2.1 (only ○ were fitted). The dashed curve is obtained with the previously determined v 0 and n = 1 (as expected if only one base was rate determining). ( d ) Replication rate versus force for Klenow: low stringency hybridization ( K = 24, J = 298; ⋄), high stringency hybridization ( K = 9, J = 114; ○). Replication rate for Klenow minus exo3′→5′ high stringency hybridization ( K = 4, J = 50; □). The full curve is a fit to ⋄, where v 0 = 13.5 b/s and n = 4.

    Techniques Used: Hybridization

    Time evolution of the replication on a pXΔII ssDNA under a tension of 1 pN (specific priming). ( a ) Number of bases N ( t ) replicated by Klenow minus exo3′→5′ as a function of time in two successive replication runs on the same template. Notice that pauses in replication occur at different positions in successive runs. The dots represents the raw data after low-pass filtering at ≈1 Hz. The extension of the DNA is converted into the number of added nucleotides N ( t ) as explained in the text. The full lines are polygon fits (see Materials and Methods ) with an average time duration of 100 s. ( b ) N ( t ) for Sequenase in two runs on different molecules. The full lines are polygon fits with an average time duration of 10 s.
    Figure Legend Snippet: Time evolution of the replication on a pXΔII ssDNA under a tension of 1 pN (specific priming). ( a ) Number of bases N ( t ) replicated by Klenow minus exo3′→5′ as a function of time in two successive replication runs on the same template. Notice that pauses in replication occur at different positions in successive runs. The dots represents the raw data after low-pass filtering at ≈1 Hz. The extension of the DNA is converted into the number of added nucleotides N ( t ) as explained in the text. The full lines are polygon fits (see Materials and Methods ) with an average time duration of 100 s. ( b ) N ( t ) for Sequenase in two runs on different molecules. The full lines are polygon fits with an average time duration of 10 s.

    Techniques Used:

    2) Product Images from "Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine"

    Article Title: Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh542

    Incorporation of c 3 dATP by three Family A polymerases [Taq, Bst and Klenow (exo – )]. 20% polyacrylamide gel. Incubations used the SS primer (5′-GCGTAATACGACTCACTATAG-3′), the T Template (3′-CGCATTATGCTGAGTGATATCTGCGCAGAG-5′) and either dATP or c 3 dATP (indicated below lane) alone for 2 min or with dATP or c 3 dATP for 2 min, followed by addition of dCTP, dGTP and TTP and an additional 2 min incubation. Unextended primer is at position N; addition of dATP or c 3 ).
    Figure Legend Snippet: Incorporation of c 3 dATP by three Family A polymerases [Taq, Bst and Klenow (exo – )]. 20% polyacrylamide gel. Incubations used the SS primer (5′-GCGTAATACGACTCACTATAG-3′), the T Template (3′-CGCATTATGCTGAGTGATATCTGCGCAGAG-5′) and either dATP or c 3 dATP (indicated below lane) alone for 2 min or with dATP or c 3 dATP for 2 min, followed by addition of dCTP, dGTP and TTP and an additional 2 min incubation. Unextended primer is at position N; addition of dATP or c 3 ).

    Techniques Used: Incubation

    ( A ) Loss of primer upon incubation with dATP, dCTP, dGTP and TTP, trap and Family A polymerases. The extension of the primer by the first nucleotide, dCTP opposite dG, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). A small loss of extended DNA is observed after the addition of each subsequent nucleotide. Due to the small amount of primer initially extended by 1 nt by Taq, very little full-length product is observed. ( B ) Loss of DNA primer extension after addition of each nucleotide with c3dATP, dCTP, dGTP and TTP. Again, extension of the primer by the first nucleotide, dGTP opposite dC, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). The relative loss of extended DNA with each subsequent nucleotide addition does not differ between polymerases.
    Figure Legend Snippet: ( A ) Loss of primer upon incubation with dATP, dCTP, dGTP and TTP, trap and Family A polymerases. The extension of the primer by the first nucleotide, dCTP opposite dG, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). A small loss of extended DNA is observed after the addition of each subsequent nucleotide. Due to the small amount of primer initially extended by 1 nt by Taq, very little full-length product is observed. ( B ) Loss of DNA primer extension after addition of each nucleotide with c3dATP, dCTP, dGTP and TTP. Again, extension of the primer by the first nucleotide, dGTP opposite dC, is observed least with Taq, more so with Bst and most with Klenow fragment (exo – ). The relative loss of extended DNA with each subsequent nucleotide addition does not differ between polymerases.

    Techniques Used: Incubation

    Related Articles

    Amplification:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min. .. This was followed by PCR amplification using a system containing the double-stranded cDNA template, 1 × Phusion High-Fidelity Buffer, 10 μM primers A 30 and B 30 (Table 1) , 0.5 U Phusion High-Fidelity DNA Polymerase (NEB, USA).

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: Libraries preparation using method 3 Method 3 for the library preparation was modified from a procedure reported previously as sequence independent single primer amplification (SISPA) (Allander et al., 2001; Cheng et al., 2010; Djikeng et al., 2008; Palacios et al., 2007) (Fig. 1) . .. The second-strand cDNA synthesis was modified using the denaturated RT system which was added with 1 μL Klenow fragment (5 U) (NEB, USA) and 2.33 μL 10 × NEBuffer, and the synthesis was performed at 37°C for 60 min and 75°C for 20 min.

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: Paragraph title: Sample amplification and labeling ... Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples.

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation). .. The dUTP-containing strands were then removed, and PCR amplification was performed with library-specific TruSeq PCR primers.

    Agarose Gel Electrophoresis:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min. .. The library amplicons around 450 bp were collected with the E-Gel ® SizeSelect™ Agarose Gel.

    Plasmid Preparation:

    Article Title: Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins
    Article Snippet: The efficiency of the digestion was monitored by adding 40 ng of phosphorylated linearized plasmid to an aliquot of each reaction tube. .. RNases were digested with Proteinase K. The ssDNA of purified SNS was converted into dsDNA: First, SNS and 2 pmol random hexamer primers (Roche) were denatured together 5 min at 100°C, then a slow annealing was achieved by cooling down the samples from 80°C to room temperature; second, the dsDNA was synthesized by using 0.17 units/µL of Klenow fragment for 1 h at 37°C; third, the fragments were ligated with 2 units/µL of Taq DNA Ligase (New England BioLabs) for 45 min at 45°C; finally, dsDNA was extracted, precipitated, resuspended in Milli-Q water, and quantified before library preparation.

    Article Title: Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.
    Article Snippet: After exhaustive digestion with Asp718 (an isoschizomer of Kpni) and Pvul (which cuts in the vector sequence), the resulting fragments were ligated to complementary fragments from the plasmid pAGP previously digested with Asp718 and Pvul. (The Asp718 site in pAGP is in the 3'end of the HIV-I polgene and corresponds to position 3707 in the sequence of Power et al. 119861 .) .. For mutants B and E, the frame had to be corrected by digesting the plasmids with Asp718 and filling in the 5'overhang using the Klenow fragment of DNA polymerase I (New England Biolabs).

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples. .. Microarray hybridization, scanning and data analysis Oligonucleotide microarrays were hybridized for 12 h at 65 • C and washed as detailed elsewhere (Bowtell and Sambrook, 2003) .

    Article Title: Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli.
    Article Snippet: Construction of tRNA anticodon-loop libraries Vector pACGFP, derived from pAC123 (Liu et al., 1997) , contains a linker derived from pGFPuv (Clontech) between unique EcoRI and PstI restriction sites¯anked by the strong lpp promoter and rrnC terminator. .. A cassette that corresponds to the sequence for tRNA Ser 2 with eight or nine random nucleotides in place of the 7 nt anticodon loop was made by extension of synthetic oligonucleotides (Genosys) with the Klenow fragment of E. coli DNA polymerase I (NEB) and inserted into pACGFP between EcoRI and PstI sites.

    Synthesized:

    Article Title: Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins
    Article Snippet: .. RNases were digested with Proteinase K. The ssDNA of purified SNS was converted into dsDNA: First, SNS and 2 pmol random hexamer primers (Roche) were denatured together 5 min at 100°C, then a slow annealing was achieved by cooling down the samples from 80°C to room temperature; second, the dsDNA was synthesized by using 0.17 units/µL of Klenow fragment for 1 h at 37°C; third, the fragments were ligated with 2 units/µL of Taq DNA Ligase (New England BioLabs) for 45 min at 45°C; finally, dsDNA was extracted, precipitated, resuspended in Milli-Q water, and quantified before library preparation. .. The same method of dsDNA conversion was applied to sheared and denatured genomic DNA to be used as sequencing control.

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: .. Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation). ..

    Isolation:

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: Briefly, poly(A) mRNAs isolated from 3 μg of total RNA using oligo(dT)25 Dynabeads (Invitrogen) were fragmented at 94°C for 5 min in buffer containing ProtoScript II reaction buffer (New England BioLabs [NEB]), hexamer (Qiagen), and oligo(dT)23 VN (NEB). .. Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation).

    Polymerase Chain Reaction:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min. .. This was followed by PCR amplification using a system containing the double-stranded cDNA template, 1 × Phusion High-Fidelity Buffer, 10 μM primers A 30 and B 30 (Table 1) , 0.5 U Phusion High-Fidelity DNA Polymerase (NEB, USA).

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: The second-strand cDNA synthesis was modified using the denaturated RT system which was added with 1 μL Klenow fragment (5 U) (NEB, USA) and 2.33 μL 10 × NEBuffer, and the synthesis was performed at 37°C for 60 min and 75°C for 20 min. .. This was followed with PCR using the primer SP (Table 1 ).

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: The reaction profile was 10 min at 25 • C, 60 min at 42 • C and 30 min at 50 • C. This was followed by 40 cycles of PCR amplification using primer EEadp (5 -GTAAGGTGCACGTAGTTG-3) and the profile: 30 s at 94 • C, 30 s at 40 • C, 30 s at 50 • C, 60 s at 72 • C. Additional 20 PCR cycles with primer EEadp were used to incorporate aminoallyl-dUTP (Fermentas, Ontario, Canada) to the samples. .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples.

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation). .. The dUTP-containing strands were then removed, and PCR amplification was performed with library-specific TruSeq PCR primers.

    Incubation:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: .. The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min. .. This was followed by PCR amplification using a system containing the double-stranded cDNA template, 1 × Phusion High-Fidelity Buffer, 10 μM primers A 30 and B 30 (Table 1) , 0.5 U Phusion High-Fidelity DNA Polymerase (NEB, USA).

    Infection:

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: Sample amplification and labeling dsRNA obtained from infected grapevines was denatured for 5 min at 95 • C and the RT reaction was carried out in a volume of 25 l containing 20 pmol of primer EErnd (5 -GTAAGGTGCACGTAGTTGNNNNNNNNN-3 ), 200 U of SuperScript II (Invitrogen), 40 U of RNaseOUT, 2.4 mM dNTP mix and 5 l of 5× RT buffer. .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples.

    Purification:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: .. The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min. .. This was followed by PCR amplification using a system containing the double-stranded cDNA template, 1 × Phusion High-Fidelity Buffer, 10 μM primers A 30 and B 30 (Table 1) , 0.5 U Phusion High-Fidelity DNA Polymerase (NEB, USA).

    Article Title: Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins
    Article Snippet: .. RNases were digested with Proteinase K. The ssDNA of purified SNS was converted into dsDNA: First, SNS and 2 pmol random hexamer primers (Roche) were denatured together 5 min at 100°C, then a slow annealing was achieved by cooling down the samples from 80°C to room temperature; second, the dsDNA was synthesized by using 0.17 units/µL of Klenow fragment for 1 h at 37°C; third, the fragments were ligated with 2 units/µL of Taq DNA Ligase (New England BioLabs) for 45 min at 45°C; finally, dsDNA was extracted, precipitated, resuspended in Milli-Q water, and quantified before library preparation. .. The same method of dsDNA conversion was applied to sheared and denatured genomic DNA to be used as sequencing control.

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: Purified products were labeled with Cy3 mono NHS ester (GE Healthcare, Buckinghamshire, UK) according to the manufacturer instructions. .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples.

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation). .. Following purification and quantification, the libraries were sequenced on an Illumina HiSeq4000 platform at the CLC Genomics and Epigenomics Core Facility at the Weill Cornell Medical College.

    Labeling:

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples. .. Microarray hybridization, scanning and data analysis Oligonucleotide microarrays were hybridized for 12 h at 65 • C and washed as detailed elsewhere (Bowtell and Sambrook, 2003) .

    Sequencing:

    Article Title: Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins
    Article Snippet: RNases were digested with Proteinase K. The ssDNA of purified SNS was converted into dsDNA: First, SNS and 2 pmol random hexamer primers (Roche) were denatured together 5 min at 100°C, then a slow annealing was achieved by cooling down the samples from 80°C to room temperature; second, the dsDNA was synthesized by using 0.17 units/µL of Klenow fragment for 1 h at 37°C; third, the fragments were ligated with 2 units/µL of Taq DNA Ligase (New England BioLabs) for 45 min at 45°C; finally, dsDNA was extracted, precipitated, resuspended in Milli-Q water, and quantified before library preparation. .. The same method of dsDNA conversion was applied to sheared and denatured genomic DNA to be used as sequencing control.

    Article Title: Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.
    Article Snippet: After exhaustive digestion with Asp718 (an isoschizomer of Kpni) and Pvul (which cuts in the vector sequence), the resulting fragments were ligated to complementary fragments from the plasmid pAGP previously digested with Asp718 and Pvul. (The Asp718 site in pAGP is in the 3'end of the HIV-I polgene and corresponds to position 3707 in the sequence of Power et al. 119861 .) .. For mutants B and E, the frame had to be corrected by digesting the plasmids with Asp718 and filling in the 5'overhang using the Klenow fragment of DNA polymerase I (New England Biolabs).

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: Libraries preparation using method 3 Method 3 for the library preparation was modified from a procedure reported previously as sequence independent single primer amplification (SISPA) (Allander et al., 2001; Cheng et al., 2010; Djikeng et al., 2008; Palacios et al., 2007) (Fig. 1) . .. The second-strand cDNA synthesis was modified using the denaturated RT system which was added with 1 μL Klenow fragment (5 U) (NEB, USA) and 2.33 μL 10 × NEBuffer, and the synthesis was performed at 37°C for 60 min and 75°C for 20 min.

    Article Title: Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli.
    Article Snippet: .. A cassette that corresponds to the sequence for tRNA Ser 2 with eight or nine random nucleotides in place of the 7 nt anticodon loop was made by extension of synthetic oligonucleotides (Genosys) with the Klenow fragment of E. coli DNA polymerase I (NEB) and inserted into pACGFP between EcoRI and PstI sites. .. Selection and library crossing Electrocompetent TOP10 cells were transformed with b-lactamase four-base codon reporter libraries to make reporter strains.

    Random Hexamer Labeling:

    Article Title: Differences in firing efficiency, chromatin, and transcription underlie the developmental plasticity of the Arabidopsis DNA replication origins
    Article Snippet: .. RNases were digested with Proteinase K. The ssDNA of purified SNS was converted into dsDNA: First, SNS and 2 pmol random hexamer primers (Roche) were denatured together 5 min at 100°C, then a slow annealing was achieved by cooling down the samples from 80°C to room temperature; second, the dsDNA was synthesized by using 0.17 units/µL of Klenow fragment for 1 h at 37°C; third, the fragments were ligated with 2 units/µL of Taq DNA Ligase (New England BioLabs) for 45 min at 45°C; finally, dsDNA was extracted, precipitated, resuspended in Milli-Q water, and quantified before library preparation. .. The same method of dsDNA conversion was applied to sheared and denatured genomic DNA to be used as sequencing control.

    other:

    Article Title: Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine
    Article Snippet: Taq, Klenow fragment, Klenow fragment (exo– ), Bst, T7, GB-D (Deep Vent), GB-D (exo– ) [Deep Vent (exo– )], Tli (Vent) and Tli (exo– ) [Vent (exo– )] polymerases were purchased from New England Biolabs.

    Construct:

    Article Title: Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region.
    Article Snippet: For mutants B and E, the frame had to be corrected by digesting the plasmids with Asp718 and filling in the 5'overhang using the Klenow fragment of DNA polymerase I (New England Biolabs). .. Construction A second set of deletion mutants was constructed by replacing the RSV sequences upstream of the Pstl site located near the end of RSV gag (position 2450 in Schwartz et al. 119833 ) with sequences from the 5' end of the GS-sAg gene.

    Modification:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: .. The second-strand cDNA synthesis was modified using the denaturated RT system which was added with 1 μL Klenow fragment (5 U) (NEB, USA) and 2.33 μL 10 × NEBuffer, and the synthesis was performed at 37°C for 60 min and 75°C for 20 min. .. This was followed with PCR using the primer SP (Table 1 ).

    RNA Sequencing Assay:

    Article Title: Transcriptional Responses of the Trichoplusia ni Midgut to Oral Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus
    Article Snippet: Paragraph title: RNA-Seq library preparation. ... Subsequently, first-strand cDNA was synthesized using a ProtoScript II kit, and second-strand synthesis was carried out with a reaction mix consisting of RNase H (NEB), the Klenow fragment of DNA polymerase I (NEB), and a deoxynucleoside triphosphate mix containing dATP, dCTP, dGTP, and dUTP (Promega Corporation).

    Injection:

    Article Title: Replication by a single DNA polymerase of a stretched single-stranded DNA
    Article Snippet: .. The capillary was then rinsed with 10 mM Tris (pH 8)/100 mM NaCl, after which Klenow fragment (Roche Molecular Biochemicals), Klenow fragment minus exo3′→5′ (New England Biolabs), or sequenase Version 2.0 (Amersham Pharmacia) was injected at ≈20 nM in the polymerization buffer. .. The hybridization stringency was assessed by using a nonspecific primer on the same molecule and checking that it did not initiate polymerization under the same conditions.

    Recombinant:

    Article Title: Detection of viromes of RNA viruses using the next generation sequencing libraries prepared by three methods.
    Article Snippet: It began with random RT reaction: 8 μL viral RNA, 1 μL 100 μM primer A 15 N 6 (Table 1), and 2 μL nuclease-free water were mixed and incubated at 72°C for 5 min. Then the RNA/primer mixture was placed on ice for at least 3 min. Then the mixture was added with 4 μL 5 × first-strand buffer, 1 μL dNTP (100 μM), 2 μL DTT (0.1 M), 1 μL RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 U/μL) and 1 μL SuperScript ® III Reverse Transcriptase (200 U/μl) (Invitrogen, USA), and incubated at 25°C for 15 min and 42°C for 30 min. .. The reaction was terminated at 70°C for 15 min. Then the reaction system was added with 1 μL RNase H (TaKaRa, Japan) and further incubated at 37°C for 20 min. After purification using DynaMag™-2 Magnet and Agencourt ® AMPure ® XP Reagent (Beckman Coulter, USA), the purified first-strand cDNA was used for the synthesis of the second-strand cDNA with primer B 15 N 6 (Table 1) at 70°C for 5 min. Then the mixture was added with 1 μL Klenow fragment (5 U) (NEB, USA), 5 μL 10 × NEBuffer 2, 2 μL dNTP (100 μM) and 1 μL DTT (0.1 M), and then incubated at 37°C for 30 min.

    Derivative Assay:

    Article Title: Expanding the genetic code: selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli.
    Article Snippet: Construction of tRNA anticodon-loop libraries Vector pACGFP, derived from pAC123 (Liu et al., 1997) , contains a linker derived from pGFPuv (Clontech) between unique EcoRI and PstI restriction sites¯anked by the strong lpp promoter and rrnC terminator. .. A cassette that corresponds to the sequence for tRNA Ser 2 with eight or nine random nucleotides in place of the 7 nt anticodon loop was made by extension of synthetic oligonucleotides (Genosys) with the Klenow fragment of E. coli DNA polymerase I (NEB) and inserted into pACGFP between EcoRI and PstI sites.

    Hybridization:

    Article Title: Replication by a single DNA polymerase of a stretched single-stranded DNA
    Article Snippet: Paragraph title: High Stringency Primer Hybridization. ... The capillary was then rinsed with 10 mM Tris (pH 8)/100 mM NaCl, after which Klenow fragment (Roche Molecular Biochemicals), Klenow fragment minus exo3′→5′ (New England Biolabs), or sequenase Version 2.0 (Amersham Pharmacia) was injected at ≈20 nM in the polymerization buffer.

    Article Title: A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.
    Article Snippet: Simultaneously, 0.5 pmol of Spike70F oligonucleotide was labeled with Cy5 mono NHS ester, and mixed with the sample previous to hybridization. .. Alternatively viral genomic libraries in pGEM-T Easy (Promega) vector were labeled with Cy3 after random priming with DNA Pol I (New England Biolabs, Ipswich, MA, USA) to incorporate aminoallyl-dUTP to the samples.

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    New England Biolabs dnase i buffer
    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using <t>DNase</t> I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Dnase I Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs restriction endonuclease fsp i
    Agarose gel electrophoresis of KS7-KS8 <t>PCR</t> products digested with <t>Fsp</t> I (A) or Hha I (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 ( stx 1c + stx 2d ; ONT:H8); 2, 3115/97 ( stx 1c + stx 2d ; O128:H2); 3, 521/99 ( stx 1c + stx 2d ; O rough :H19); 4, 4756/98 ( stx 1c + stx 2d ; O70:H − ); 5, 273/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 6, 295/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 7, EDL 933 ( stx 1 + stx 2 ; O157:H7); 8, 2544/00 ( stx 1 ; O145:H − ); 9, 3385/00 ( stx 1 + stx 2 ; O111:H − ); 10, 4424/99 ( stx 1 + stx 2 ; O157:H7); 11, 2049/98 ( stx 1 + stx 2c ; O157:H − ); 12, 2050/98 ( stx 1 + stx 2 + stx 2c ; O157:H − ).
    Restriction Endonuclease Fsp I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t7 endonuclease i
    Generation of  Slx2  knockout mice. A) Schematic of the genomic target sites in the  Slx2  gene. B) Analysis of CRISPR/Cas9 system efficiency in mouse embryonic stem (ES) cells by using T7E1 assay. pX330 ligated with different gRNAs were transfected into V6.5 ES cells. Targeted region was PCR amplified and then digested by T7 Endonuclease I. M, marker. NC, negative control, mouse ES cells transfected with GFP plasmid. C) gRNA-1 and gRNA-3 worked in mouse embryos. Sequencing results of mouse embryos injected with Cas9 mRNA and  Slx2  gRNA-1 or gRNA-3 were shown. D) Genotyping results of line 1 and line 11 F1 mice. Cas9-mediated indels lead to frame shift of  Slx2 . Blue color showed the sequence of gRNAs; protospacer adjacent motifs (PAM) were labeled with purple color; red color showed the modified sequences information after targeting. -, nucleic acid base deletion. E) Western blot analysis of SLX2 expression level in testis from line 1 (#1) and line 11 (#11) mice. WT, wild-type.
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 380 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Journal: Nucleic Acids Research

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    doi: 10.1093/nar/gkr051

    Figure Lengend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Article Snippet: Pellets were suspended in 5 µl DNase I buffer and 0.2 U DNase I (NEB) and 0.2 U snake venom phosphodiesterase (Worthington) was added to the reactions.

    Techniques: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Modification

    Agarose gel electrophoresis of KS7-KS8 PCR products digested with Fsp I (A) or Hha I (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 ( stx 1c + stx 2d ; ONT:H8); 2, 3115/97 ( stx 1c + stx 2d ; O128:H2); 3, 521/99 ( stx 1c + stx 2d ; O rough :H19); 4, 4756/98 ( stx 1c + stx 2d ; O70:H − ); 5, 273/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 6, 295/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 7, EDL 933 ( stx 1 + stx 2 ; O157:H7); 8, 2544/00 ( stx 1 ; O145:H − ); 9, 3385/00 ( stx 1 + stx 2 ; O111:H − ); 10, 4424/99 ( stx 1 + stx 2 ; O157:H7); 11, 2049/98 ( stx 1 + stx 2c ; O157:H − ); 12, 2050/98 ( stx 1 + stx 2 + stx 2c ; O157:H − ).

    Journal: Journal of Clinical Microbiology

    Article Title: Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans

    doi: 10.1128/JCM.40.4.1441-1446.2002

    Figure Lengend Snippet: Agarose gel electrophoresis of KS7-KS8 PCR products digested with Fsp I (A) or Hha I (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 ( stx 1c + stx 2d ; ONT:H8); 2, 3115/97 ( stx 1c + stx 2d ; O128:H2); 3, 521/99 ( stx 1c + stx 2d ; O rough :H19); 4, 4756/98 ( stx 1c + stx 2d ; O70:H − ); 5, 273/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 6, 295/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 7, EDL 933 ( stx 1 + stx 2 ; O157:H7); 8, 2544/00 ( stx 1 ; O145:H − ); 9, 3385/00 ( stx 1 + stx 2 ; O111:H − ); 10, 4424/99 ( stx 1 + stx 2 ; O157:H7); 11, 2049/98 ( stx 1 + stx 2c ; O157:H − ); 12, 2050/98 ( stx 1 + stx 2 + stx 2c ; O157:H − ).

    Article Snippet: The stxB 1 gene was amplified with primers KS7-KS8 (Table ), and 12 μl of each PCR product was digested with restriction endonuclease Fsp I or Hha I (New England BioLabs GmbH, Frankfurt, Germany), as recommended by the manufacturer.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    Generation of  Slx2  knockout mice. A) Schematic of the genomic target sites in the  Slx2  gene. B) Analysis of CRISPR/Cas9 system efficiency in mouse embryonic stem (ES) cells by using T7E1 assay. pX330 ligated with different gRNAs were transfected into V6.5 ES cells. Targeted region was PCR amplified and then digested by T7 Endonuclease I. M, marker. NC, negative control, mouse ES cells transfected with GFP plasmid. C) gRNA-1 and gRNA-3 worked in mouse embryos. Sequencing results of mouse embryos injected with Cas9 mRNA and  Slx2  gRNA-1 or gRNA-3 were shown. D) Genotyping results of line 1 and line 11 F1 mice. Cas9-mediated indels lead to frame shift of  Slx2 . Blue color showed the sequence of gRNAs; protospacer adjacent motifs (PAM) were labeled with purple color; red color showed the modified sequences information after targeting. -, nucleic acid base deletion. E) Western blot analysis of SLX2 expression level in testis from line 1 (#1) and line 11 (#11) mice. WT, wild-type.

    Journal: PLoS ONE

    Article Title: CRISPR/Cas9 Promotes Functional Study of Testis Specific X-Linked Gene In Vivo

    doi: 10.1371/journal.pone.0143148

    Figure Lengend Snippet: Generation of Slx2 knockout mice. A) Schematic of the genomic target sites in the Slx2 gene. B) Analysis of CRISPR/Cas9 system efficiency in mouse embryonic stem (ES) cells by using T7E1 assay. pX330 ligated with different gRNAs were transfected into V6.5 ES cells. Targeted region was PCR amplified and then digested by T7 Endonuclease I. M, marker. NC, negative control, mouse ES cells transfected with GFP plasmid. C) gRNA-1 and gRNA-3 worked in mouse embryos. Sequencing results of mouse embryos injected with Cas9 mRNA and Slx2 gRNA-1 or gRNA-3 were shown. D) Genotyping results of line 1 and line 11 F1 mice. Cas9-mediated indels lead to frame shift of Slx2 . Blue color showed the sequence of gRNAs; protospacer adjacent motifs (PAM) were labeled with purple color; red color showed the modified sequences information after targeting. -, nucleic acid base deletion. E) Western blot analysis of SLX2 expression level in testis from line 1 (#1) and line 11 (#11) mice. WT, wild-type.

    Article Snippet: Reaction mixture was first heated at 95°C for 5 min. 35 cycles were then carried out with the following parameters: denaturing at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 40 s. Reaction was finished with a final extension at 72°C for 5 min. PCR products were then denatured at 95°C for 5 min, annealed by cooling down at room temperature for 30 min, and treated with T7 endonuclease I (NEB, M0302L) at 37°C for 15 min. Digested DNA was separated on a 3% agarose gel.

    Techniques: Knock-Out, Mouse Assay, CRISPR, Transfection, Polymerase Chain Reaction, Amplification, Marker, Negative Control, Plasmid Preparation, Sequencing, Injection, Labeling, Modification, Western Blot, Expressing

    Detection of heterozygous mutant workers produced by artificial insemination. ( A ) Electrophoretic patterns of PCR products around the sgRNA target site in Experiments 3 and 5 for semen collection. Each PCR product was amplified from genomic DNA extracted from a hind leg of a sexually matured drone, and then treated with T7 endonuclease I. Magenta and blue arrowheads indicate the expected band size of the PCR products digested by T7 endonuclease I at the sgRNA target site (#3 and #10 in Experiment 3, #6 in Experiment 5) and at repeated A region, respectively. ( B ) Sequences around the sgRNA target site of mutant drones in ( A ). Orange dashes indicate detected deletions. ( C ) Overlapping sequences, which were obtained from direct sequencing of putative heterozygous mutant workers inferred from analysis using T7 endonuclease I, were separated using CRISP-ID. Heterozygous workers were derived from a wild-type queen artificially inseminated with semen pool 2 (containing semen of drone #10). Overlapping waveforms are seen at the right of the yellow bar in the upper panel. Separated sequences are shown below.

    Journal: Scientific Reports

    Article Title: mKast is dispensable for normal development and sexual maturation of the male European honeybee

    doi: 10.1038/s41598-018-30380-2

    Figure Lengend Snippet: Detection of heterozygous mutant workers produced by artificial insemination. ( A ) Electrophoretic patterns of PCR products around the sgRNA target site in Experiments 3 and 5 for semen collection. Each PCR product was amplified from genomic DNA extracted from a hind leg of a sexually matured drone, and then treated with T7 endonuclease I. Magenta and blue arrowheads indicate the expected band size of the PCR products digested by T7 endonuclease I at the sgRNA target site (#3 and #10 in Experiment 3, #6 in Experiment 5) and at repeated A region, respectively. ( B ) Sequences around the sgRNA target site of mutant drones in ( A ). Orange dashes indicate detected deletions. ( C ) Overlapping sequences, which were obtained from direct sequencing of putative heterozygous mutant workers inferred from analysis using T7 endonuclease I, were separated using CRISP-ID. Heterozygous workers were derived from a wild-type queen artificially inseminated with semen pool 2 (containing semen of drone #10). Overlapping waveforms are seen at the right of the yellow bar in the upper panel. Separated sequences are shown below.

    Article Snippet: The PCR products were then annealed, and treated with T7 endonuclease I (New England Biolabs, Japan) according to manufacturer’s protocol (T7EI assay).

    Techniques: Mutagenesis, Produced, Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay