direct plasmid sequencing problems - direct plasmid sequencing problem (Feb/28/2006 )
well this is my problem.
I need to sequence directly from a vector but the results I got back are really bad. Before sending the sequences away we performed a PCR to check for contaminations, these where not present in the negative controls and the fragments amplified look realy nice. Then how come I can't sequence with these primers directly from the plasmid?
thanks for your help....
I'm not sure what you mean -- how do you check for contaminants with PCR?
There is no reason that primers that perform well in PCR won't also work when sequencing -- sequencing is, after all, PCR. Did you send the right amount of template DNA and primer for sequencing?
Have you considered sequencing the PCR product, if you're having no luck with sequencing from the plasmid?
are there any priming sites on the vector that you can use?
in most commercially available vectors there are priming sites, that flank the cloning site. take a look at the vector map and try sequencing with that primers (sometimes included in the cloning kit or commercially available). Then you can also see, if the insert has been built in correctly.
If you are just interested in the sequence of your insert, sequencing of your pcr product will do as well!
You don't tell us what is "bad." An example would help a lot. A common problem is too much template DNA, which ironically makes the reaction fail. But we can't tell without more information.
Your primer may be incompatible with the sequencing chemistry or cycler program (ie annealing temp). Try using a standard primer flanking your insert. If your insert is too long to sequence completely with the standard primer then you can use your results to design a better sequencing primer.