I forgot treat my samples with proteinase K before the DNA extraction step - (Mar/19/2012 )
Hi everybody! I hope you can help me. When I was performing a Chip I made a mistake and I forgot treat my samples with proteinase K (PK), after the reverse-crosslinking step. I mean, after the overnight reverse-crosslinking step, I extracted my DNA with phenol-cloroform and just after this step I realized I forgot put the proteinase K. Then I treated my samples with proteinase and I performed another extraction with phenol-cloroform and finally I carried out the precipitation with glycogen and ethanol. My question is: Is necessary and very important to treat my samples with PK, BEFORE the DNA-extraction step? That can be the cause to obtain a low yield of my DNA recovery?
Hard to say. I'd just go with it and do the PCR.........as long as all your samples (including the Input) were trated the same it really shouldn't matter. The only thing is that since you lose DNA during the isolation step, and you performed this step twice, it might reduce your final DNA yeild........but if your doing real-time PCR then you should be able to detect everything at some Ct..........even if it is later than you'd normally expect