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