Klenow

Manufactured by New England Biolabs

Sourced in United States

Klenow is a large fragment of DNA Polymerase I from E. coli, which retains the 5' to 3' polymerase activity but lacks the 5' to 3' exonuclease activity. It is commonly used for a variety of DNA manipulation applications, such as DNA labeling, nick translation, and fill-in reactions.

Automatically generated - may contain errors

Lab products found in correlation

Kapa hyperplus kit, by Roche (4 mentions) Odyssey clx imager, by LI COR (3 mentions) Tnt sp6 quick coupled transcription translation system, by Promega (3 mentions) Xbai, by New England Biolabs (3 mentions) T4 dna ligase, by New England Biolabs (3 mentions) Superscript 3, by Thermo Fisher Scientific (3 mentions) Superscript 4, by Thermo Fisher Scientific (2 mentions) Ecori, by New England Biolabs (2 mentions) α 32p dctp, by PerkinElmer (2 mentions) Bamhi, by New England Biolabs (2 mentions) Miseq reagent kit v3, by Illumina (2 mentions) Random hexamer, by Thermo Fisher Scientific (2 mentions) Phosphoimager, by Fujifilm (1 mentions) Dynabeads m 280, by Thermo Fisher Scientific (1 mentions) Reticulocyte lysate, by Promega (1 mentions) Random primer, by Thermo Fisher Scientific (1 mentions) Miseq v3 flow cell, by Illumina (1 mentions) Pbabe puro, by Addgene (1 mentions) Wizard sv gel and pcr clean up system, by Promega (1 mentions) Agei hf, by New England Biolabs (1 mentions)

28 protocols using klenow

DNA Barcoding of Transcription Factors

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A step-by-step protocol describing the assignment of DNA barcodes to TF proteins can be found at Protocol Exchange⁵⁷. To assign a unique DNA barcode to each protein, a collection of 2000 address oligos (Integrated DNA Technologies) were synthesized, each of which containing a BsaI recognition site and a cutting site (GGTCTCCGACT) at the 5′-end, a 8 nt random sequence as unique molecular identifiers (UMI), a 7–11 nt unique DNA barcode sequence, and a 20-nt consistent sequence, complementary to the anchor oligo (Supplementary Fig. 1a and Supplementary Table 1). Next, the address oligos were individually annealed to the anchor oligos conjugated on the TF proteins in a 96-well format, followed by a Klenow polymerase reaction (1 × NEB Buffer 2 with 0.6 mM dNTP mix, 1 U Klenow [New England Biolabs], 12 uM address oligos) at 37 °C for 30 min to synthesize the complementary strands. Free address oligos were removed with three washes in the same washing buffer described above. 1/50 of the bead slurry of each protein was taken from two adjacent plates (i.e., 192 = 2 × 96) and pooled. A total of eight protein pools were generated for the DAPPL reactions.

Song G., Wang G., Luo X., Cheng Y., Song Q., Wan J., Moore C., Song H., Jin P., Qian J, & Zhu H. (2021). An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine. Nature Communications, 12, 795.

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Chromatin Digestion and End Repair

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After chromatin digestion and reaction stop, washing twice with NicE-C wash buffer (2 mM MgCl₂, 1× BSA, 2.5% PEG8000, 0.05% SDS, 0.2% Triton X-100), the nuclei were resuspended in 400 μL 1 × T4 ligase buffer containing 20 μL of 10% Triton X-100, 20 μL of 50% PEG8000, 10 μL of 10 mM dNTPs, 10 μL of T4 Polynucleotide Kinase (NEB, M0201S), 8 μL of T4 DNA Polymerase (NEB M0203S), 2 μL of Klenow (NEB M0210S). Then, the nuclei were incubated for 60 min at 37°C for end repair with interval shaking (950 rpm, 15 sec every 2 min). Five hundred millimolar EDTA was added to the final 30 mM and incubated for 20 min at 65°C to stop the reaction. After washing twice with NicE-C wash buffer, the nuclei were resuspended in 400 μL 1× NEBuffer2 containing 20 μL of 10% Triton X-100, 20 μL of 50% PEG8000, 10 μL of 10 mM dATP, 10 μL of Klenow (exo-) (NEB M0212S). Then, the nuclei were incubated for 60 min at 37°C for dA tailing with interval shaking (950 rpm, 15 sec every 2 min). Five hundred millimolar EDTA was added to the final 30 mM and incubated for 20 min at 65°C to stop the reaction.

Luo Z., Zhang R., Hu T., Zhu Y., Wu Y., Li W., Zhang Z., Yao X., Liang H, & Song X. (2022). NicE-C efficiently reveals open chromatin–associated chromosome interactions at high resolution. Genome Research, 32(3), 534-544.

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Biotinylated DNA Beads for Ku80 Binding

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DNA beads were prepared as previously described (Postow et al., 2008 (link)). Briefly, pBluescript SK+ vector was cut and filled with Klenow (New England Biolabs), biotin-dATP and biotin-dUTP (Chemcyte) and bound to M280 dynabeads (Invitrogen). 25–50 μl extract was used for every 1 μg of DNA on beads. ³⁵S-labelled Ku80 was generated in reticulocyte lysate (Promega) using X. laevis Ku80 cDNA (Postow et al., 2008 (link)) and diluted 1:10 in egg extract. Beads were removed from extract with a magnetic particle separator and eluted by denaturation. For Ku80 release assays, Ku80 was quantified using a phosphoimager (Fujifilm).

van den Boom J., Wolf M., Weimann L., Schulze N., Li F., Kaschani F., Riemer A., Zierhut C., Kaiser M., Iliakis G., Funabiki H, & Meyer H. (2016). VCP/p97 extracts sterically trapped Ku70/80 rings from DNA in double strand break repair. Molecular cell, 64(1), 189-198.

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Electrophoretic Mobility Shift Assay for Zebrafish NR2F1A

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EMSA was performed essentially as previously reported [77 (link)]. Oligonucleotides were designed containing the nr2f1a DR1 site (GTGTCAAAGTTCA), the nr2f1a DR1 site with a targeted mutation in the second half site of the DR1 abolishing the direct repeat (GTGTCAAAGTCAT), and a previously reported Cyp26a1 DR5 site [76 (link)]. A complementary oligonucleotide was designed with a 5’ LI-COR IRDye 700 (IDT). The oligonucleotides were annealed and the ends filled with Klenow (New England Biolabs). Zebrafish myc-rarab was in the pCS2+MT. Zebrafish RXRba was cloned into pCS2p+. Proteins for EMSA were made using the TnT SP6 Quick Coupled Transcription/Translation System (Promega). Protein samples were gently mixed with LI-COR tagged probes and incubated at room temperature for 20 minutes. 4% polyacrylamide gels were run for 2 hours at 150 V. Gels were imaged using an Odyssey CLx LI-COR imager.

Dohn T.E., Ravisankar P., Tirera F.T., Martin K.E., Gafranek J.T., Duong T.B., VanDyke T.L., Touvron M., Barske L.A., Crump J.G, & Waxman J.S. (2019). Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors. PLoS Genetics, 15(2), e1007962.

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Targeted Viral Genome Sequencing

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cDNA was made using random primers (Thermo Fisher) and SuperScript IV (Thermo Fisher) and dsDNA was made using Klenow (NEBNext). The KAPA Hyper Plus kit (Roche) was used to prepare the library for sequencing with minor adjustments: fragmentation time was reduced to 3 min and the adapters were diluted 1:10. Targeted enrichment was performed using VirCapSeq [22 (link)] and all 50 samples were pooled equimolarly and sequenced on an Illumina MiSeq v3 flow cell (2 × 300 bp).

Embregts C.W., Farag E.A., Bansal D., Boter M., van der Linden A., Vaes V.P., van Middelkoop-van den Berg I., IJpelaar J., Ziglam H., Coyle P.V., Ibrahim I., Mohran K.A., Alrajhi M.M., Islam M.M., Abdeen R., Al-Zeyara A.A., Younis N.M., Al-Romaihi H.E., AlThani M.H., Sikkema R.S., Koopmans M.P., Oude Munnink B.B, & GeurtsvanKessel C.H. (2022). Rabies Virus Populations in Humans and Mice Show Minor Inter-Host Variability within Various Central Nervous System Regions and Peripheral Tissues. Viruses, 14(12), 2661.

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Isolation and integration of TCRs

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Previously published 6132A cancer-specific CD8⁺ T cell clones (anti-6132A-A1, [12 (link)]) and cancer-specific CD4⁺ T cell clones (anti-mRPL9, [13 (link)]) were used for TCR gene isolation. TCRs were determined by 5’-RACE-PCR following manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA) and were integrated into pMP71-PRE as described [18 (link)] (pMP71-anti-6132A-A1, -anti-mRPL9). For selection of TCR-positive T cells, pMP71-anti-mRPL9-IRES-GFP or pMP71-anti-6132A-A1-IRES-mCherry were generated [19 (link)]. In brief, pMP71-TCR plasmids were digested with EcoRI (NEB, Ipswich, MA, USA) and NotI (NEB), followed by incubation with Klenow (NEB) to create TCR blunt end fragments. The pMP71-IRES-GFP or pMP71-IRES-mCherry vectors were digested with PmlI (NEB) followed by dephosphorylation with CLIP (NEB). TCR fragments and opened pMP71-IRES-GFP or pMP71-IRES-mCherry vector were ligated to generate color-linked TCR expression vectors pMP71-anti-A1-IRES-mCherry and pMP71-anti-mL9-IRES-GFP.

Diehl M.I., Wolf S.P., Bindokas V.P, & Schreiber H. (2020). Automated cell cluster analysis provides insight into multi-cell-type interactions between immune cells and their targets. Experimental cell research, 393(2), 112014.

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Electrophoretic Mobility Shift Assays with Zebrafish Transcription Factors

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EMSAs were performed as described previously [129 (link)]. Oligonucleotides were designed containing the ripply3 DR1 site (AGGTCAGAGGTCA), the ripply3 DR4 (AGTTCCTCAGGGGTCA) site, and a previously reported Cyp26a1 DR5 site [130 (link)]. A complementary oligonucleotide was designed with a 5’ LI-COR IRDye 700 (IDT). Sequences for oligos are listed in S1 Table. The oligonucleotides were annealed and the ends filled with Klenow (New England Biolabs). Zebrafish myc-rarab was cloned into pCS2+MT. Zebrafish RXRba was cloned into pCS2p+. Proteins for EMSA were made using the TnT SP6 Quick Coupled Transcription/Translation System (Promega). Protein samples were gently mixed with LI-COR tagged probes and incubated at room temperature for 20 minutes. 4% polyacrylamide gels were run for 2 hours at 150 V. Gels were imaged using an Odyssey CLx LI-COR imager.

Song Y.C., Dohn T.E., Rydeen A.B., Nechiporuk A.V, & Waxman J.S. (2019). HDAC1-mediated repression of the retinoic acid-responsive gene ripply3 promotes second heart field development. PLoS Genetics, 15(5), e1008165.

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EMSA with Modified Oligonucleotides and Klenow

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EMSA was performed essentially as reported in [70 (link)], with the following modifications. Target oligonucleotides were designed with a 15 bp 3’ (ACATTCGCGCAGATC) extension. A common complementary oligonucleotide to the 15 bp extension was synthesized with a 5’ LI-COR IRDye 700 (IDT). The oligonucleotides were annealed and the ends filled with Klenow (New England Biolabs). Proteins for EMSA were made using the TnT SP6 Quick Coupled Transcription/Translation System (Promega). Gels were imaged using an Odyssey CLx LI-COR imager.

D’Aniello E., Ravisankar P, & Waxman J.S. (2015). Rdh10a Provides a Conserved Critical Step in the Synthesis of Retinoic Acid during Zebrafish Embryogenesis. PLoS ONE, 10(9), e0138588.

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Lentiviral GFP-MUC4/X Plasmid Construction

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Lentiviral GFP‐MUC4/X plasmid (pLenti‐GFP‐MUC4/X) was constructed by inserting the cDNA of GFP‐MUC4/X at the EcoRV site of pLAS5w.Pbsd vector. GFP‐MUC4/X cDNA was obtained by digesting pCMV3‐MUC4‐C‐GFPSpark (Sino Biological #HG16066‐ACG) with KpnI (NEB) and XbaI (NEB) and filling the sticky end with Klenow (NEB). pLAS5w.Pbsd vector was obtained from National RNAi Core Facility (Taipei, Taiwan). The retroviral pBABE‐puro (#1764; RRID: Addgene_1764) plasmid and retroviral pBabe‐Kras G12D‐puro plasmid (#58 902; RRID: Addgene_58 902), encoding HA‐KRASG12D, were acquired from Addgene (USA). The lentiviral shRNA expression vectors of pLKO.1‐shLacZ (TRCN0000072223), shInhba #1 (TRCN0000067740) and shInhba #2 (TRCN0000324943) were from the National RNAi Core Facility (Taipei, Taiwan).

Hu C., Huang C., Hsu M., Chien H., Wu P., Chen Y., Jeng Y., Tang S., Chung M., Shen C., Chang M., Chang Y., Tien Y, & Lee W. (2023). Oncogenic KRAS, Mucin 4, and Activin A‐Mediated Fibroblast Activation Cooperate for PanIN Initiation. Advanced Science, 10(36), 2301240.

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Profiling Antibody Repertoire Diversity

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The technique for end-labeling Ig fragments to observe the spectrum of CDR3 lengths in an Ig pool was described for Xenopus H chain [32 ] and shark L chain [33 ]. Briefly, purified PCR products were digested with Ava II in a 10 ul volume for one hour, after which 1 ul from a 2.5 ul mixture of α-³²P-dCTP (15 µCi, PerkinElmer) and Klenow (5 units, New England Biolabs) was added. After 15 minutes at room temperature, 5 ul of STOP buffer was added. The samples were denatured and loaded onto a 4% acrylamide-urea sequencing gel together with a control sequencing reaction using M13mp18 phage to calibrate strand size. The PCR reaction, using the NS4L and JL5A primers (Supplemental Table IA), generated fragments of ~380 bp. Digestion of the Ava II site in FR3 produced fragments of 265 bp and the varying bands of <120 bp containing CDR3.

Iacoangeli A., Lui A., Naik U., Ohta Y., Flajnik M, & Hsu E. (2015). Biased immunoglobulin light chain gene usage in the shark. Journal of immunology (Baltimore, Md. : 1950), 195(8), 3992-4000.

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