FAQ: What are the expected results using the included control?

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.  


Example of a control reaction for the EnGen Mutation Detection Kit

Gel depicting a control reaction for the EnGen Mutation Detection Kit, comparing unpurified, undigested to T7 Endonuclease I-digested PCR product
Lane 1 is the Quick-Load Purple 1 kb Plus DNA Ladder (NEB #N0550). Lanes 2 and 3 are replicate samples (R1 and R2) of unpurified, undigested PCR product (600bp). Lanes 4 and 5 are replicates (R1 and R2) of T7 Endo I digested PCR products.