PCR question

Jody K. Hirsh jkh141 at nwu.edu
Mon May 8 09:37:02 EST 1995


In article <9505051952.AA23370 at oak.cats.ohiou.edu>, aa374885 at OAK.CATS.OHIOU.EDU (Ahmet Arman) says:
>
>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.
>
>
>A. Arman
>aa374885 at oak.cats.ohiou.edu
Did you check w/ software to see that your primers are not binding to another site with a similar Tm?
Also, have you done controls eliminating primers or templates? Did you get the sharp band 
previously and then it started to get smeary?  Templates and primers can get wierd after
multiple freeze-thaws (check the archives for this).
Sometimes I believe there are gel artifacts from ss and ds dna. Try denaturing and renaturing the product, or do this as a 
slow ramp to 4 degrees from 95 at the end of the run.  Also check that your thermocycler is 
behaving --check printouts, make sure its not doing any wierd fluxes in temp during a run. You may need to watch and time several cycles 
because sometimes the printouts only tell you the temp at the end of the temp step, and if something strange happened during that segment it would not 
get recorded.
Also, you may need to increase extension? What polymerase are you using? Pfu and maybe vent are less processive 
and need 3-5X longer extension times than Taq.

Jody K. Hirsh
Northwestern University, Evanston, IL.   USA
jkh141 at nwu.edu



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