PCR on a bacterial pellet

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ssgoj
ssgoj's picture
PCR on a bacterial pellet

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?

mnmolecular
mnmolecular's picture
I've PCR'd from bacterial

I've PCR'd from bacterial several times and found that less is better.  Remember how sensitive PCR is and use perhaps 100X less genomic DNA than you've tried and see if you get better results.

Maybe try completely suspending a single overnight colony in 1mL of water and then use 2uL in your PCR.  I don't know which Taq polymerase you're using but I've had very good results with KOD Hot Start.  I have no connection with the manufacturer.  

If you're seeing the genomic DNA in your agarose I would say you are using dramatically more genomic than you should.

Best of luck.