3 replies to this topic

[AGM1]

Hi,

I have an issue with the PCR I am running. The expected PCR product is 1169 base pairs long, but I keep getting a band >10,0000 bp when I run the pcr reaction mix on a 1% gel. I have attached a picture, which shows 4 samples. All have the large band, but one (& its 1/100 dilution) has a band of the desired size. The DNA template is purified fosmid (basically very large low copy plasmid) DNA, which is about 48,000 bp long. The DNA is also from a pooled culture, so multiple clones are present in the extracted DNA. The goal is that if a pool PCRs as postivie, then at least one clone in the pool may be positive and a subsequent PCR on that clone can be performed. I am trying to PCR a gene that is about 1500 bp that may be present on the insert. Does anybody have any suggestions as to what the large band that I am observing is? Could it be the fosmid DNA, some strange non specific PCR product, etc?

Thanks


Gel annotation:

Wells are on the left half of the gel.
The top sample is a positive control at 1169 bp.
Followed by the four samples, and a 1 KB ladder is on the bottom well.

Attached Thumbnails

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Edited by AGM1, 23 June 2011 - 03:51 PM.

[phage434]

I think those bands are your template.  Ordinary gels don't distinguish 20Kb from 50Kb from 200 Kb bands.  The ease with which you can see them indicates to me that you have far too much template DNA in your reaction.  Try repeating with 100x and 1000x dilutions of your template.

[leelee]

+1

[Ameya P]

AGM1,

Like Phage said, there is far too much DNA in your reaction. Also, you figured that out when you used the 100 fold dilution, and got the specific product, you were looking for. So to answer your doubts, the large bands is your template DNA and the one where you get the PCR product is the pool, where you clone lies.

Good luck with your cloning!