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1、T7E1 protocol 1. CRISPR Transfection. 4872h 2. Genomic DNA prep. 3. Target site PCR. 4. Denature/Reannealing 5. T7 endonuclease I reaction. 6. Agarose gel eletrophoresis T7E1 protocol 1. CRISPR Transfection. 4872h Methods and conditions for transfection varies for each cell lines. Use the optimized
2、transfection protocol (reagents, DNA amount, Etc.) for your cell line ! Caution Optimal transfection efficiency (at least 50%) is critical. - Nucleofection seems to be most broadly applicable. T7E1 protocol 4872h 2. Genomic DNA prep. Genomic DNA prep at 48-72 hours post-transfection using any commer
3、cial or in-house genomic DNA prep protocol/kit T7E1 protocol 4872h 3. Target site PCR. Most important step! Robust and clean PCR amplification is critical. Thus, nested PCR (not required but recommended) is useful. We use Primer3 program for primer design (http:/frodo.wi.mit.edu/primer3/) * 1st PCR
4、: 6001kb size around Target site * 2nd PCR : 400500bp size around Target site - Size above is what we use but not essential Nested PCR - 1st PCR :20cycle - Use small amount (for example, 0.1ul) for 2nd PCR (35 cycle) Analyze PCR product on agarose gel to confirm that the PCR products are abundant an
5、d that no non-specific products appear. T7E1 protocol 4872h 4. Denature/Reannealing Wt/wt hybrid, wt/mut hetero Denature/annealing protocol.(PCR cycle design) 1. 95 2min 2. -2 /s to 85 . 3. -0.1 /s to 25 . 4. 16 forever. The reaction mixture can be stored in 4 degree for 12 days T7E1 protocol 4872h
6、5. T7 endonuclease I reaction. Use 10 ul (0.5-1ug) of reaction mixture after Denature/annealing step for mismatch-sensitive nuclease assay. - The amount of reaction mixture may need optimization for different PCR reactions Mismatch-sensitive nuclease used for detection of EN- induced mutation is T7
7、endonuclease 1 (T7E1) from NEB or Surveyor nuclease from Transgenomics. - below are the protocol for assay using T7E1. Please refer to the protocol from manufacturer for surveyor assay. T7E1 assay condition. : 20min 37 incubation * Total 20ul * 10x NEB #2 buffer 2ul PCR product 10ul T7E1 enzyme 0.25
8、ul DW up to 20ul - Reaction time and amount of T7E1 enzyme may need optimization. Access time or T7E1 activity cause non-specific degradation of DNA (smearing signal on agarose gel) 6. Agarose gel eletrophoresis T7E1: mismatch digest T7E1 protocol Use 22.5% agarose gel. It may be needed to detect the progression of PCR and cut product bands to determine optimal running time Make sure to run same amount of substrate DNA (DNA after denature/annealing step) without T7E1 enzyme treatment as a negative control 6. Agarose gel eletrophoresis
