PCR on whole yeast cells

Anthony Hilton A.C.Hilton at bham.ac.uk
Wed Jun 8 03:45:34 EST 1994


In article <2sumhu$spf at mercury.dur.ac.uk>, Bert Popping
<Bert.Popping at durham.ac.uk> wrote:

> Dear Alvin,
> 
> I haven't found any reference in a journal, but there is a summary of this 
> topic in the archive. I used this technique last week and I got excellent 
> results! The reason why you can't find it in the literature is propbably 
> because it's just to simple. 
> 
> But here comes the summary:
> 
> 
> Let me summarize our current experience:
> 
> 1.  As many of you have said, THERE IS NO NEED TO ISOLATE DNA.  We have  
> found that one toothpick's worth (we use the small end of a disposable  
> innoculating loop to avoid contamination), or about 1/2 of a 2 day YPD  
> colony, resuspended directly in a 100 microliter PCR reaction, works just  
> fine.  There is also no need to preboil; just resuspend and go.  
> 
> 2.  We have also found that the PCR product is cuttable by at least one  
> restriction enzyme (Sal1) DIRECTLY from the tube.  Kind of surprising with  
> all those yeast guts swimming around, but there you have it.  Just spin the  
> tube to pellet the glop and use the supernatant.
> 
> Needless to say, with this kind of ease of use, whole new worlds have been  
> opened up to us in terms of genetic screens.  What a great technique.  The  
> guy who invented this should get some sort of prize, at least.
> 
> 
> --
>                                               ... usual disclaimer applies.
> ________________________________________
>  	Dr. B. Popping				                          
>  	University of Durham			    			  
>  	Dept. Biol. Sci. 	  PHONE   +44 91 374 2430 	          
>  	South Road                FAX     +44 91 374 2417 	          
>  	Durham DH1 3LE	          E-MAIL  Bert.Popping at durham.ac.uk       
>  	ENGLAND			  
> ________________________________________
> 	WWW set pointer to:   http://www.dur.ac.uk/~dbl1bp	  


I have tried to perform whole cell PCR on isolates of Salmonella and
Campylobacter using 5ul of an overnight culture in Luria broth as the
source of DNA. Even with pre-boiling I still can't seem to get the
fragments amplified.

Any tips for bacterial wholw cell PCR?

Thankee

Wig.
> ________________________________________				



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