Smeared PCR products

G G
Tue Feb 28 13:18:59 EST 1995


In article <3idj6b$2pd at ccshst05.cs.uoguelph.ca>, cjerome at uoguelph.ca (Cheryl C Jerome) says:
>
>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
Check your DNA on an agarose gel to see if it's degraded
also your primers may have gone off (were they stored at -20'?)
If your -ve is positive then either your lab area is contaminated or more 
likely, your primers are



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