New England Biolabs, E3321S, EnGen® Mutation Detection Kit

Related Categories Validation of CRISPR-based Gene Editing,, DNA Repair Enzymes and Structure-specific Endonucleases Specification Materials Required but not Supplied Oligodeoxyribonucleotide primers for PCR PCR reaction tubes or strips Nuclease-free water Thermocycler Aerosol tips for PCR FAQ Q: Why do I see an extra band when I run the undigested heteroduplex on an agarose gel? A: In some cases undigested heteroduplex DNA will run slightly slower than the homoduplex DNA. Depending on the composition of the mutations this can sometimes be seen as a band running above the homoduplex on an agarose gel. Q: Why do I see very little DNA on my gel or low signal on the fragment analyzer? A: Verify that the yield of the PCR reaction was sufficient before proceeding with digestion and analysis. Perform the control reaction to make sure that the kit is performing correctly. Q: Will EnGen® T7 Endonuclease I recognize single base mismatches and single base insertions or deletions (indels)? A: T7 Endonuclease does not efficiently recognize indels or mismatches less than two bases. Q: Can cell lysates be used in the PCR reaction? A: We have successfully tested crude extract prepared using QuickExtract™ (Epicentre #QE09050) and DNAzol® Direct (Molecular Research Center, INC #DN 131). We cannot recommend the use of other reagents. Additional PCR reagents are included for optimization of the amplification. Q: What are the expected results using the included control? A: The included Control Template and Primer mix contains two plasmids that when amplified will produce amplicons with and without a 10 bp insertion. The primers included in this mix will amplify a ~600 bp region around this insertion. After denaturation and re-annealing of these PCR products, some will form heteroduplexes. The T7 Endonuclease I will detect the heteroduplexes and cut them to yield fragments of ~200 bp and ~400 bp. The fragments can be detected by standard agarose gel electrophoresis or by using fragment analyzers. Q: My PCR reaction yield is low, can I add more than 5 µl of the PCR reaction to the digestion reaction? A: The digestion has been optimized for use with up to 5 µl of unpurified PCR reaction. Adding more than 5 µl of the PCR reaction could interfere with the digestion reaction. We recommend optimizing the PCR reaction to get a higher yield or concentrating the PCR reaction by column purification using NEB Monarch PCR & DNA Cleanup Kit (5 μg) (NEB #T1030). Q: There is a precipitate in the bottom of the Master Mix tube? Is this normal? A: Q5® Master Mix may precipitate upon freezing and thawing. This does not indicate a problem with the product. For optimal performance, thaw fully and resuspend any precipitate by warming to room temperature and inverting gently until completely dissolved.