Smeared PCR products

Chris Barry chbarry at ucdavis.edu
Sun Feb 26 22:33:41 EST 1995


cjerome at uoguelph.ca (Cheryl C Jerome) wrote:
>
> Hello.  I am wondering if anyone can help me.  I have been having PCR 
> problems for a couple of months.  I have been having consistent smearing 
> with my PCR amplified product.  A bright smear starts at my PCR product 
> and extends to my primers.  Since my PCR product is large (2.8 KB) it is 
> a very large smear which extends to my PCR band.  The smearing is also 
> the negative control.  I have replaced my Taq, my primers, dNTPs, etc... 
> to try to figure out what the problem is.  Also, I am running a positive 
> control with DNA which has worked well in the past.  I had the PCR 
> working with the same conditions and primers for about 2 months.  Then, I 
> was sequencing for about a month and was not running PCR --- when I 
> started to run PCR again, I had the smears and have not been able to 
> resolve the problem.  I have re-autoclaved water, etc....Is it possible 
> there is something in my water source?  what causes smearing? I would 
> appreaciate any suggestions. Thank you. Cheryl

Since they are large fragments, have you tried to play around with the
magnesium concentration of the buffer or to extend the polymerization
cycle of PCR (Give it more time to extend before you denature it again).

                                        Good Luck




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