PCR from yeast genomic DNA

Michael Hamilton mhamilto at gpu.srv.ualberta.ca
Mon Jan 8 04:28:05 EST 1996


Jon Stewart <jds2 at ufcc.ufl.edu> wrote:

>I need to PCR amplify two Saccharomyces genes of approx. 1000 bp, 
>neither of which contains an intron according to the sequence database.  
>Since I have never attempted PCR from yeast genomic DNA, I have some 
>questions.  Is it possible to use the S. cerevisiae genomic DNA (> 100 
>kb) sold by Promega ($95 / 100 ug) for this purpose?  My other simple 
>choice is the Clontech Quick cDNA ($310 / 20 reactions).  Obviously, if 
>the Promega genomic DNA will work, I can find other uses for the $200 
>price difference :-).  In addition, any hints or special tricks for 
>running these PCR reactions would be greatly appreciated.

>Jon Stewart
>Department of Chemistry
>University of Florida


	You don't need the clontech kit for PCR.  Saccharomyces has a very
stripped down genome and false primings are low if you pay attention
to your primer selection.
	Don't you have access to a culture of Saccharomyces? I find that I
don't need very large DNA fragments at all for my preps.  for PCR up
to 2.5 KB I have good results with the following:  

DNA isolation

	A one ml overnight culture (YEPD) is resuspended in 100 ul of 3% SDS,
TE for 15 min (RT).  add 400 ul TE and resuspend.  Extract once with
500 ul phenol-chloroform.  Remove 400 ul to new tube, and EtOH
precipitate.  dry pellet on benchtop until translucent, and then
resuspend in 50-100 ul TE.  yeilds about 1-2 ug DNA (~20 kb avg) This
is not my protocol, I have the refernce somewhere....

	If you think you need something cleaner and larger (50-100 KB) use the
protocol in Methods in Enzymology vol 192 (?).  


PCR

rxn mix  

1-5 ul of DNA 
10 ul 10x dNTP mix (2.5 um ea)
10 ul BRL PCR buffer
10 ul 10x primer 1
10 ul 10x primer 2 
3units Taq
water to 100 ul

Cycling (Robocycler from Stratagene)

4 min 94 C --denaturation

30s 99 C
1 min 62 C        Cycle
1min/kb 72 C

5min at 72 C to finish off fragments.  The times are based on a paper
by Wayne Barnes PNAS 1994 I know I have the reference around here
somewhere.......

Geneclean reaction to isolate fragment, and then process for
subcloning.  (polish ends, restrict, etc)

Other tips

primer selection:  Since saccharomyces has a high AT content, a primer
with a >45 % GC content is very specific.  Stay away from primers with
poly-A runs since these are common intervening sequences (IMHO)

	The actual amount of DNA used is quite flexible, and generally not as
sensitive to secondary annealing sites as mammalian or drosophila DNA.
Because of the small genome size, 10 ng still gives you a good
starting copy number.    I often don't need more than 25 cycles to get
enough product for three sequencing runs. (course, I'm using 100-200
ng starting material) 

	using DNA which is isolated via the longer, kinder method, I've been
able to get up to 15 kb fragments with the klenTaq1/Pfu system (No, I
don't know who's marketing it now, it used to be a company called AB
peptides)

	Also, most of the "naughty" Taq in use is apparently lacking in the
3'-5' exo activity (proofreading)  if you are concerned about
fidelity, use one of the proofreading polymerase varieties.  


Hope this helps, Mikey.  
\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/\/||||+
Michael Hamilton                      Dept of Biol Sci
oughta-be PhD                         U of Alberta
                                      Edmonton, Alberta T6G 2E9
Ph    403-492-1103                    Canada
FAX   403-492-1903




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