PCR smear after one-month storage - (Dec/26/2011 )
I am trying to PCR amplify a 1.5kb human gene from a pET28a plasmid vector containing it. The first time I tried about a month ago, it worked fine. The plasmid was miniprepped from a high copy producing strain of E coli. In 50ul of reaction, I used 50-100ng of plasmids, primers (10uM), buffers, and NEB phusion polymerase,
Recently, I took the same one-month old plasmid containing colony that I stored in 4C, and did the PCR reaction under same conditions. The result was a SMEAR above the 1.5kb PCR product. (Please see attached picture if it's helpful)
Can plasmids get degraded (in either elution buffer or within cells) during that one-month storage period? I don't think primer dimers formed during the reaction because they should appear to be smaller than 1.5kb.
I also suspect that the my primers, which I ordered a month ago, may have been degraded over time.
Would thawing and replating a new bunch of cells or ordering new primers improve the quality of my PCR products?
Thanks in advance
It looks to me as if your PCR worked. The smear may be an overloaded gel rather than a failed PCR. Storing DNA in water at 4C is ill-advised. Store it (and in my opinion elute it) in TE, where it will be much much more stable. Plasmids can also be lost in storage at 4C. I would definitely change my habits of storage if I wanted long term access to your constructs.
Thank you for your reply. I stored both the plasmids and primers in TE buffers at -20C.
I suppose I can use less because the recommended PCR template was 10ng instead of 50 or 100.