I have a PCR product made of 2 pieces. I am doing site direct mutagenesis of an enzyme. I use PCR to create the 2 parts. I purify the 2 parts using a cleanup kit. I then run PCR a second time to assemble to the 2 parts and amplify them.
When I run on a gel so that I can cut out the appropriate band and purify for further processing, it is stuck in the well. I see some slight smearing down the lane, but most of it is in the well. There is no evidence of the 2 parts that were used as there are no bands present for the parts.
The combined gene is about 1200 bp, the parts are about 950 and 250 bp. I use a 1% gel. The 260/280 ratios were 1.86 and 1.87 for the 2 PCR parts used. I am using Bullseye Taq, but never had a problem like this in the past.
I have searched and most say it is a protein or some other contamination. But being a purified PCR products and primers, I don't see this as the case.
Could I have overloaded the well with too much product?
Any thoughts or suggestions would be appreciated.
is it possible that there is a homologous region of the 2 fragments that allows multiple concatenation thus a very long product formation from the 2 parts. The very long product would move slowly from the well. This could be tested by pcr of primers at the ends of the 2 pieces...if it produced a ladder of too long amplimers that could be the problem . Possibly restriction digest of your 2nd pcr product ( unpurified ) might provide a clue also if the wrong sizes were produced
Mutagenesis part aside that I really did not get, you have PCR amplified two fragments that you are trying to assemble using SOE PCR? Before doing a preparative electrophoresis to excise out the 1200 bp amplicon, did you run a small volume of PCR reaction to confirm if your SOE PCR actually worked?
Thank you for your answers.
This is an issue that has just showed up. We have never had a problem until the past 2 weeks. My student had it it happen to 2 of his samples, but not the other 2 on the same gel.
It has happened twice on my samples now. We have 2 pieces with a 21 bp overlap region. We then use these 2 parts as the template and add in the forward and reverse primers to complete the gene. The PCR product is then run on a gel to excise just the complete gene for purification. Normally we would see some residual of 1 or both of the parts.
Could it be too much DNA and the gel is getting clogged? Because all that you see is a bright well and a light smear. There are no bands for the parts or the gene.
I did not run as small volume on the gel. That was my next step in troubleshooting. I skipped it initially as we either see the band if it worked or just the parts if it didn't. I know it shouldn't be a Taq issue or a contaminant issue as others have had.
Could it have produced too much DNA for the gel to handle?
dont worry about the smears the smears are rather degraded pcr products that occurs in the process of down stream processing as u do pcr and then combined pcr, the amplicon become template twice so some amplicons degrade, so dont worry about the smears, secondly i would say u decrease the start conc of amplicon PCR to 5 ng in combined pcr
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