dadler@milton.u.washington.edu (David Adler) (01/09/91)
Raimo Pollanen writes; Has anyone made PCR from tissue or cell samples without extracting DNA by phenol-chlorophorm precipitations ... I have done a few PCR's on remnants of amniotic fluid samples and it seemed to work fine. Of course you want to make sure cells are lysed - freezing and thawing thru several cycles should work - this is in fact what happened to amniotic fluid before I got it. I do not know how many cells but it was fractions of an original 1 ml of amniotic fluid, so not many. I amplified a repetitive target, maybe 1000 copies per cell, as well as a single copy sequence.
pollanen@csc.fi (01/09/91)
Hello again! Has anyone made PCR from tissue or cell samples without extracting DNA by phenol-chlorophorm precipitations And how were the results like if you compared them to those PCR results done by extracted DNA material? I have tried to make PCR from cervical swabs samples to find HPV DNA. But there has been some difficulties Yours Raimo Pollanen(M.Sc.) Pollanen@CSC.FI
WEH%FDACFSAN@PUCC.PRINCETON.EDU (01/10/91)
We've had good success with some bacterial cells by simply boiling for a minute or two. Depending on how you want to do it, a couple of microliters of this *boilate* can be added directly to a PCR tube or you can try precipitating with ethanol. You can also lyse cells with guanidine isothiocyanate and then ethanol precipitate.
bertheau@jouy.inra.fr (Yves Bertheau) (01/12/91)
But did you use this boiling method (or guanidine isothiocyanate method ) with
samples contaminated by polysaccharides (e.g. plant polysaccaharides).
Generally speaking do you know a method for the extraction of DNA from
infected plants ?
Thanks
Yves Bertheau
INRA Plant Pathology
PARIS FRANCEWEH%FDACFSAN@PUCC.PRINCETON.EDU (01/15/91)
Sorry, we've only used it with oysters and milk. We did use some plasmid extraction methods with lettuce but these are cumbersome, requiring typical phenol-chloroform extractions. If you keep the sample amount low (that is, don't try to make too concentrated of a preparation) heating with guanidine isothiocyanate (maybe to 60C or even boiling) and following with an ethanol precipitation (just add sodium acetate to 0.3M) essentially ignoring the guanidine may yield satisfactory results. Good luck and let me know if you are successful.