Klenow exo polymerase

Manufactured by New England Biolabs

Klenow exo-polymerase is a DNA polymerase enzyme derived from the Klenow fragment of E. coli DNA Polymerase I. It possesses 5' to 3' polymerase activity but lacks the 5' to 3' exonuclease activity of the full-length DNA Polymerase I enzyme.

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Lab products found in correlation

Genepix 4000b, by Molecular Devices (1 mentions) Bs seq, by Diagenode (1 mentions) Proteinase k, by New England Biolabs (1 mentions)

Close spelling variants of this product

Klenow fragment (3′–5′ exo-) polymerase (KF polymerase) Klenow exo-DNA polymerase Klenow exo- Klenow polymerase

2 protocols using klenow exo polymerase

Array-based Comparative Genomic Hybridization

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Array-based comparative genomic hybridization (aCGH) was performed in biological duplicate on CRB, LEP, and MUSH relative to a DBY8268 control as previously described (Pollack et al. 1999 (link)). Samples were labeled using amino-allyl dUTP (Ambion), Klenow exo-polymerase (New England Biolabs), and random hexamers, and coupled with cyanine dyes (Amersham). Samples were hybridized to custom 385K tiling arrays (NimbleGen) designed using chipD (Dufour et al. 2010 (link)) on the composite S. cerevisiae genome described above. Arrays were hybridized in a NimbleGen hybridization system 12 (BioMicro) and scanned using a scanning laser (GenePix 4000B, Molecular Devices) according to NimbleGen protocols (http://www.nimblegen.com/, last accessed September 15, 2014). Data normalization was performed using Bioconductor (Gentleman et al. 2004 (link)) and custom Perl scripts. The affy() package (Gautier et al. 2004 (link)) was used to apply probe-level quantile normalization to the log₂ ratios. We defined genes with increased copy number as those with a log₂ aCGH ratio greater than 0.7 (because relative intensity values are often slightly compressed from the expected duplication log₂ value of 1.0); genes with a log₂ aCGH ratio < −1.0 were identified as potentially deleted. All microarray data are available through the NCBI Gene Expression Omnibus under the accession GSE56441.

Wohlbach D.J., Rovinskiy N., Lewis J.A., Sardi M., Schackwitz W.S., Martin J.A., Deshpande S., Daum C.G., Lipzen A., Sato T.K, & Gasch A.P. (2014). Comparative Genomics of Saccharomyces cerevisiae Natural Isolates for Bioenergy Production. Genome Biology and Evolution, 6(9), 2557-2566.

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Enzymatic Conversion of 5mC to 5pyC

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10 ng of gDNA ligated to 5mC- or 5pyC-containing adapters was used as input. A methylated copy-strand was created. First, 1 μM of copy primer was annealed (Supplementary Table 1, v1), in a total volume of 10 μL in CutSmart Buffer and 1 mM final concentration individually of dATP/dGTP/dTTP (Promega) and dmCTP (NEB). 5 units of Klenow (exo-) polymerase (NEB) was then added and incubated for 30 min at 37°C. After purification (Zymo Oligo Clean & Concentrator), libraries were mixed with 1 μM untagged M.MpeI-N374K and 160 μM CxSAM in carboxymethylation buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated for 1 hour at 37°C followed by denaturation for 5 min at 95°C. 1 μL of Proteinase K was added (NEB) and samples were incubated at 37°C for 15 min. The samples were purified using SPRI beads (1.2x) and eluted in 1 mM Tris-Cl, pH 8.0. DNA was then subjected to BS-Seq (Diagenode, see below) or to snap-cooling and A3A deamination in a final volume of 50 μL as previously described ^{25 (link)}. Purified DNA was then amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA) before purification over SPRI beads (0.8X) to yield final libraries. The non-optimized workflow was used in Figure 2.

Wang T., Fowler J.M., Liu L., Loo C.E., Luo M., Schutsky E.K., Berríos K.N., DeNizio J.E., Dvorak A., Downey N., Montermoso S., Pingul B.Y., Nasrallah M., Gosal W.S., Wu H, & Kohli R.M. (2023). Direct enzymatic sequencing of 5-methylcytosine at single-base resolution. Nature chemical biology, 19(8), 1004-1012.

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