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Hi, this is my first post in molecularstation.
I am trying to PCR amplify a 1.5kb sequence from a pET28a plasmid vector containing my gene of interest. The first time I tried about a month ago, it worked fine. Please see attached picture.
However, when I tried again with the same plasmid (50-100ng), primers (10uM), buffers, and NEB phusion polymerase a few days ago, there was a smear tailing my PCR product. See other attached picture.
I grew the plasmid-bearing E coli cells in a drug media and miniprepped it, and I diluted the 100uM primers to 10uM and used those as well. But the smear appeared again.
All PCR reactions were 50ul and I loaded 10ul to each well. I suspect that the my primers, which I ordered a month ago, became degraded, or my plasmids went "bad" over the one-month period.
Has anyone else experienced this problem before and how may I fix this problem? Thanks
Re: PCR Smear
According to your first picture, there was a slight almost invisible smear that can be observed. This smear became more prominent when you reran your PCR this time.
I can't say for sure the cause - but it's unlikely that your primers degrade as primers are generally very stable. Your plasmids should be ok, if you don't freeze-thaw it often (I'd suggest keeping it in aliquots if you use them often).
The smear could indicate that you're using too much template. What I would suggest is you reduce the amount of template used. Vortex/shake the template before pipetting it into the reaction tube - I'd suggest trying 10x-100x dilution. Next, do a gradient temperature (e.g. 0.3 C increments).
Try this two approaches, first separately and then together. If it still doesn't work...well make a new post here.
Re: PCR Smear
Yes primers and plasmids are usually stable for many years when frozen, so I don't think that's the problem.
If the primers or the plasmid were the problem, your smear should appear all over. But now it's only above the band, which looks to me like the problem is in the gel. Maybe the gel is simply overloaded, your PCR reaction was actually too efficient! You could try loading a smaller amount of the reaction.
Also, the first gel looks generally "neater", i.e. the bands are sharper and starighter. I also experinence this kind of variability which usually doesn't make any practical difference. But I would anyway make sure that the gel is fresh and the buffer too next time, just to be on the saft side.
|pcr , smear|
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