PCR question

Eric C. Anderson anderson at pharmdec.wustl.edu
Sun May 7 15:01:42 EST 1995


In article <9505051952.AA23370 at oak.cats.ohiou.edu>,
aa374885 at OAK.CATS.OHIOU.EDU (Ahmet Arman) wrote:

> Hi netters!
> I am doing PCR using 60 bp template and 15 mer primers. My PCR conditions:
>   94 oC 1 min
>   54 oC 1 min
>   72 oC 1 min
> 
> I never get single sharp band and am getting smear DNA. I checked all possible
> things like change annealing temperature, reduce template and time period of
> denaturation and extention which affect my PCR however; I ever get
single 60 bp sharp band. Is
> there anybody which has better idea to get single sharp band.
> 
> every suggestion will be appreciated.

i'm confused here.  is your template a 60bp piece or are you trying to PCR
a 60bp piece.  and from what kind of template, genomic, cDNA, plasmid?

you might try titrating the Mg2+ concentration as yet another variable. 
if all that you're trying to get is a 60bp fragment, i would think that
much shorter incubation times might also help.

good luck,
eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)362-7058 (FAX)
660 s. euclid box 8103                           
st. louis, mo 63110

for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html



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