Lba Cas12a (Cpf1) Enzyme酶 BioVector NTCC质粒载体菌种细胞基因保藏中心

Lba Cas12a (Cpf1) Enzyme酶 BioVector NTCC质粒载体菌种细胞基因保藏中心
Lba Cas12a (Cpf1) from Lachnospiraceae bacterium ND2006 is a site-specific DNA endonuclease guided by a single 41-44 nucleotide guide RNA (gRNA) (1). Targeting requires a gRNA complementary to the target site as well as a 5´ TTTN protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. Cleavage by Lba Cas12a occurs ~18 bases 3´ of the PAM and leaves 5 nucleotide 5´ overhanging ends. Lba Cas12a has Simian virus 40 (SV40) T antigen nuclear localization sequences (NLS) at both the N and C-termini of the protein.  Required Materials:  Lba Cas12a (Cpf1) (BioVector-M0653) 10X BioVectoruffer 2.1 Reaction Buffer Nuclease-free water Proteinase K, Molecular Biology Grade (BioVector-P8107) Guide RNA containing the targeting sequence in the region of interest DNA substrate containing the target sequence The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides Optional Materials: Apparatus and reagents for DNA fragment analysis oAgarose gel electrophoresis apparatus DNA Loading Dye (e.g., Gel Loading Dye, Purple (6X) (BioVector-B7024S) oAgilent Bioanalyzer or similar Before You Start: We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found here. Reactions are typically 30 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes. It is essential to keep the molar ratio of Cas12a and gRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found here. Prepare 300 nM gRNA by diluting the stock with nuclease-free water on ice. Prepare 30 nM substrate DNA with a single target sequence by diluting the stock with nuclease-free water on ice. If planning to use higher concentration Lba Cas12a (BioVector-M0653T) for in vitro digestion of DNA, the enzyme can be diluted to 1 μM in 1X Buffer 2.1 (BioVector-B7202) prior to the reaction assembly and used immediately. If the 1 μM dilution will be stored at -20°C, it should be diluted using the enzyme storage buffer: 500 mM NaCl, 20 mM sodium acetate, 0.1 mM EDTA, 0.1 mM TCEP and 50% glycerol (pH 6.0 @ 25°C). Procedure: 1.Assemble the reaction at room temperature in the following order*: COMPONENT  AMOUNT Nuclease-free water   20 µl BioVectoruffer 2.1 Reaction Buffer (10X)   3 µl 300 nM gRNA   3 µl (30 nM final)  1 µM Lba Cas12a (Cpf1)   1 µl (~30 nM final)  Total Reaction Volume  27 µl 2.Pre-incubate for 10 minutes at 25⁰C. 3.Add 3 µl of 30 nM substrate DNA (30 µl final volume). 4.Mix thoroughly and pulse-spin in a microfuge. 5.Incubate at 37°C for 10 minutes. 6.Add 1 µl of Proteinase K (BioVector-P8107) to each sample, Mix thoroughly and pulse-spin in a microfuge.  7.Incubate at room temperature for 10 minutes. 8.Proceed with analysis.  References: 1.Zetsche, B., et. al. (2015) Cel,l 163, 759-771.
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