Hi there,
I am performing a PCR-reaction on a bacterial pellet and getting no results. The pellet was freeze-dried en then resuspended in 500 microliters of LB-medium.
Is it a problem that i'm performing it on the pellet? I think it doesn't matter because the cells will burst open at 95 degrees en the gDNA will be free in the PCR mix.
this is my PCR mix:
Cell Culture 2 (10x diluted) 1 1.5 2 FWD primer (10 µM) 5 5 5 5 RVD primer (10 µM) 5 5 5 5 2 mM dNTP’s 5 5 5 5 10x pfu pol buffer 5 5 5 5 10xdiluted pfu 1.25 1.25 1.25 1.25 ddH20 26.75 27.75 27.25 26.75 Totale 50 50 50 50
And my PCR program is:
5 minutes - 95 degrees
30 sec- 95 degrees 30x
30-60 sec - 50-58 degrees 30x
7 min- 72 degrees 30x
10 min - 72 degrees
After a agarose-gel is see the gDNA (i think) in the slots and a smear in the lanes but no bands. I also have a positive control so the components ( dNTP's, POLymerase buffer, polymerase) are working.
Does someone have a solution for this problem? Or need i to do a DNA extraction because the cells do not lyse fast enough