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,

"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

More information about the Methods mailing list