2 replies to this topic

[qpwoei4756]

Hi,

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

qp

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[phage434]

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.

[qpwoei4756]

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.