PCR on Genomic DNA from cell lysate - (Apr/02/2008 )

Hi,
Does anyone have a protocol for preparation of cell lysate for PCR on the genomic DNA of the cells?
Can this be done at all? We want to perform PCR on the genomic DNA without the need to extract the DNA.

Thanks,
NGY

-NGY-

Just lysing cells will often work. Maybe have a look at some in situ PCR protocols. With in situ PCR, you can even perform PCR on tissue samples on slides.

-wbla3335-

What cell are you trying to amplify? Human cell line, bacteria? yeast? or fungal?

I have done this method using bacteria cell when i screen my clone varians using E. Coli cell.
What I did was, I culture my bacteria in LB, put in 1.5ml tube, spin down the pellete, discard supernatant,
boiled the pellet in water bath (90-95oC) for 10min --> this will lyse the bacteria cell.
Then I spin down pellet (bacteria debris) and transfer the supernatant (contain bacteria DNA) to another
fresh tube --> AND DO PCR AT THE SAME DAY.

THIS METHOD IS ONLY GOOD FOR SCREENING. THE DNA EXTRACTED IS NOT PURE AND CAN NOT STORE LONG.
YOU WILL SEE DEGRADATION WITHIN A DAY OR TWO.

-Hadrian-

QUOTE (Hadrian @ Apr 4 2008, 10:00 PM)

Thanks for your answer, however we are trying to do it on mamalian cell line. After PCR we got all material stuck in the well of the gel, so I was afraid the DNA was not detached from histones etc. Any idea?

Thanks again.
NGY

-NGY-

QUOTE (NGY @ Apr 2 2008, 07:51 AM)

Treat the cells with tritonx100 and protenaseK, then boil for 10 minutes. You may observe gDNA degradation, but its ok for pcr.

-WHR-

since you mention that your PCR product stuck in the well of the gel....

WHR is correct.. mamalian cell line will have histone protein attached to the DNA. so treat with proteinase K.

Also, I am thinking of that you have too much of gDNA (>1ug) in your PCR reaction.
Did you quatitate it before your PCR?

-Hadrian-