We have been trying to amplify our 2kb plasmid in the E.coli strain dh5a. The colony PCR after transformation (using primers targeting a 412bp region of the plasmid) yielded perfect sized bands. we then preformed plasmid miniprep and the resultant plasmid was amplified using the same primer set, but they showed multiple bands, in addition to the 412bp. Initially 2ul of template was used for a concentration of 5ng of plasmid but lowering the template concentration to 1ul, still yielded multiple bands.
The plasmid needs to have a perfect band for it to be sequenced
how to resolve this issue?
Did you use the same PCR conditions for the colony PCR and for the PCR performed on the plasmid prep ? How does your undigested plasmid prep look on an agarose gel ? One posibility could be that your plasmid forms concatemers, which would explain why you get bands larger than 412 bp upon amplification. However, even so, the 412 bp band should be present iin large excess over higher Mw bands.
Given the low cost of sequencing, I would still suggest that you try to have the plasmid sequenced. With one read starting from each side of the insert, you should get a better view of what is going on. Otherwuse, you could also retransform your plasmid prep, isolate tranformants and extract the plasmid again. If PCR still gives spurious bands not compatible with concatemers (see above), I would suspct some contamination of your PCR reagents.
it is possible that 5 ng of plasmid was not sufficient for PCR amplication, in case the plasmid quality extracted was not good. How about to try with high concentration of plasmid DNA ?
Why all the PCR?
A simple restriction enzyme analysis will show you whether vector and insert have the respective size and whether the quality is o.k. (no visible smear or visble RNA).
For sequencing, you may use the plasmid and ideally use a primer derived from the vector.
You could cut out your band and gel purify. Also, run the template adjacent to the amplicon on a gel to see if the contaminating band is template. These guys answered a lot of my questions regarding this topic: https://www.yourbiohelper.com/
What kind of polymerase did you use for colony PCR and PCR from purified plasmids? If the reactions conditions are similar, then plasmid concentration may be an issue. Usually purified plasmid DNA of 50-100ng/50ul reaction mix with 25 cycles gives good amplified band. Adding 1ul DMSO in your mix can reduce the problems of high GC content, if that is your case.
And, obviously, cross check your results through restriction digestion and sequencing.
Try to increase the annealing temperature that would help in controlling non-specific amplifications.
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