Enrichment PCR

[yuguh]

Enrichment PCR

• Quote

Post by yuguh » Wed Aug 17, 2011 2:12 pm

I am doing an enrichment PCR (40 cycles) of a restriction digest product purified with Qiagen PCR clean up kit. I get no band in my experimental lanes, correct band in my positive control and a correct band in my NTC. I have designed different primers switched up all my reagents and I still get the same thing. If it were contamination I would expect bands in my experimental lanes as well. WHAT IS GOING ON!

[canalon]

• Quote

Post by canalon » Wed Aug 17, 2011 3:34 pm

Purify your positive control with the same kit (after mixing it with the same buffers and stuff minus the restriction enzymes) and see if it still amplify. It will tell you if your purification procedure work or not.
Before that check what you are eluting DNA with (no EDTA, just 10mM Tris).