Modifying Enzymes
Modifying Enzymes
New England Biolabs, Inc. Exonuclease I Reaction Buffer – 6 ml
This is the NEB reaction buffer supplied with Exonuclease I. New England Biolabs supplies a 10X reaction buffer with all of its enzymes. At a 1X concentration this reaction buffer assures optimal activity of the enzyme.
New England Biolabs, Inc. Thermostable USER III Enzyme - 50 units
Thermostable USER (Uracil-Specific Excision Reagent) III Enzyme generates a single nucleotide gap at the location of a uracil. Thermostable USER III Enzyme is a mixture of Afu Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease IV. Afu UDG catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease IV breaks the phosphodiester backbone at the 3’ and 5’ sides of the abasic site so that base-free deoxyribose is released leaving a 5’ deoxyribose phosphate (dRP) and a 3’-hydroxyl. Thermostable USER III Enzyme also has 3’-diesterase activity which can remove 3’ phosphate, 3’-alpha, beta-unsaturated aldehyde, phosphoglycoaldehyde, and other 3' blocking groups. Thermostable USER III Enzyme is optimally active at 65C.
New England Biolabs, Inc. EnGen Spy Cas9 HF1 - 500 pmol
EnGen Spy Cas9 HF1 is a high-fidelity, quadruple substitution (N497A/R661A/Q695A/Q926A) variant of EnGen Spy Cas9 NLS from Streptococcus pyogenes with reduced non-specific DNA cleavage. Spy Cas9 is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. The single guide RNA (sgRNA) targets Cas9 to the region immediately upstream of a 5'-NGG-3' protospacer adjacent motif (PAM) producing a double stranded break 3 bases upstream of the PAM. EnGen Spy Cas9 HF1 contains Simian virus 40 (SV40) T antigen nuclear localization sequence (NLS) on the N- and C-termini of the protein.
New England Biolabs, Inc. T7 DNA Ligase – 750000 units
T7 DNA Ligase is an ATP-dependent ds DNA ligase from bacteriophage T7. It will catalyze the formation of a phosphodiester bond between adjacent 5' phosphate and 3' hydroxyl groups of duplex DNA. Cohesive end ligation and nick sealing can be efficiently catalyzed by T7 DNA Ligase. However, unlike T4 and T3 DNA Ligases, blunt end ligation is not efficiently catalyzed by T7 DNA Ligase. Addition of high concentrations of PEG 6000 [>= 20% (w/v)] to the reaction can force T7 DNA Ligase to have measurable activity. However, under typical reaction conditions blunt-end DNA ligation does not occur in the presence of T7 DNA Ligase, making it a good choice for applications in which blunt and cohesive ends of DNA are present but only the cohesive ends are to be joined. T7 DNA Ligase does not ligate blunt-end DNA fragments.
New England Biolabs, Inc. T4 RNA Ligase 2, truncated KQ – 2000 units
T4 RNA Ligase 2, truncated KQ (T4 Rnl2tr R55K, K227Q) specifically ligates the preadenylated 5' end of DNA or RNA to the 3' OH end of RNA. The enzyme does not use ATP for ligation but requires pre-adenylated linkers. T4 Rnl2tr R55K, K227Q is a double-point mutant of T4 RNA Ligase 2, truncated . Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation. K227Q reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr, by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5-phosphates of input RNAs. Mutation of R55K in T4 Rnl2tr K227Q increases the ligation activity of the enzyme to levels similar to T4 Rnl2tr. The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for high-throughput sequencing library construction of small RNA .
New England Biolabs, Inc. RNase HII – 1250 units
Ribonuclease HII (RNase HII) is an endoribonuclease that preferentially nicks 5' to a ribonucleotide within the context of a DNA duplex. The enzyme leaves 5' phosphate and 3' hydroxyl ends. RNase HII will also nick at multiple sites along the RNA portion of an Okazaki fragment.
New England Biolabs, Inc. Hi-T4™ DNA Ligase – 20000 units
A thermotolerant variant of T4 DNA Ligase, Hi-T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5 phosphate and 3 hydroxyl termini in duplex DNA or RNA and is designed to function at higher temperatures than wild type T4 DNA Ligase. Hi-T4 DNA Ligase will join blunt end and cohesive end termini as well as repair single-stranded nicks in duplex DNA, RNA or DNA/RNA hybrids at temperatures as high as 50C.
New England Biolabs, Inc. EpiMark Methylated DNA Enrichment Kit – 25 reactions
The EpiMark Methylated DNA Enrichment Kit will selectively bind and enrich double-stranded methyl-CpG DNA from fragmented genomic DNA with as little as 5 ng of input DNA. Methylated DNA is isolated from fragmented genomic DNA (5 ng-25 ug) by binding to the methyl-CpG binding domain of human MBD2 protein fused to the Fc tail of human IgG1 (MBD2-Fc), which is coupled to paramagnetic hydrophilic protein A beads (MBC2-Fc/Protein A Magnetic Bead). Two Fc domains can be bound to one site on protein A with high affinity (Kd=10-7). As the Fc fragment is a dimer, four MBD2 domains are exposed to the solvent per molecule of protein A, increasing the relative equilibrium constant 100-fold. This stable complex will selectively bind double-stranded methylated CpG containing DNA. After simple wash steps followed by magnetic capture, the enriched DNA sample is easily eluted in a small volume of nuclease-free water by incubation at 65C.
New England Biolabs, Inc. Cas9 Nuclease, S. pyogenes – 400 pmol
Cas9 Nuclease, S. pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif) . The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence.
New England Biolabs, Inc. α1-2,4,6 Fucosidase O - 80 units
a1-2,4,6 Fucosidase O is a broad specificity exoglycosidase that catalyzes the hydrolysis of terminal a1-2, a1-4 and a1-6 linked fucose residues from oligosaccharides. a1-2,4,6 Fucosidase O cleaves a1-6 fucose residues more efficiently than other linkages.
New England Biolabs, Inc. EnGen Lba Cas12a (Cpf1) – 70 pmol
EnGen Lba Cas12a from Lachnospiraceae bacterium ND2006 is a site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA) . Targeting requires a gRNA complementary to the target site as well as a 5' TTTV protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Cleavage by EnGen Lba Cas12a occurs ~18 bases 3' of the PAM and leaves 5' overhanging ends. EnGen Lba Cas12a has Simian virus 40 (SV40) T antigen nuclear localization sequences (NLS) at both the N and C-termini of the protein.
New England Biolabs, Inc. EnGen Lba Cas12a (Cpf1) – 2000 pmol
EnGen Lba Cas12a from Lachnospiraceae bacterium ND2006 is a site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA) . Targeting requires a gRNA complementary to the target site as well as a 5' TTTV protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Cleavage by EnGen Lba Cas12a occurs ~18 bases 3' of the PAM and leaves 5' overhanging ends. EnGen Lba Cas12a has Simian virus 40 (SV40) T antigen nuclear localization sequences (NLS) at both the N and C-termini of the protein.
New England Biolabs, Inc. AfeI – 200 units
A restriction endonuclease that recognizes the sequence AGC_^GCT.
New England Biolabs, Inc. PspXI – 200 units
A restriction endonuclease that recognizes the sequence VC^TCGA_GB.
New England Biolabs, Inc. Endoproteinase AspN – 50 µg
Endoproteinase AspN (flavastacin) is a zinc metalloendopeptidase which selectively cleaves protein and peptide bonds N-terminal to aspartic acid residues.