I am having problems sequencing an insert:
I've started from a commercial pcDNA3.1 plasmid with a target protein gene coupled to a truncated fluorescent protein gene (YFP). This plasmid has been amplified by maxiprep and verified by sequencing to be the expected sequence. Also, the plasmid has been digested and religated to replace the truncated fluorescent protein sequence with the full protein sequence. Bacteria were then transformed with the religated plasmid and minipreps were performed to select colonies which contained the insert (verified by PCR). When trying to sequence this plasmid with the same primers used in the PCR there was no signal of any kind on any of the several occasions when sequencing was attempted (also by using different primers at the same concentrations). The insert sequence is GC rich and that has been considered throughout the protocol and sequencing. I've been trying to sequence ~100 ng/uL plasmids using 1 nM primers.
However, when transfecting HEK293T cells with this plasmid, I observed both by flow cytometry and confocal microscopy YFP fluorescence (YFP is supposed to be bound to the protein of interest) that several days post-transfection was mainly located in cytosolic aggregates (this may be due to overexpression). It should be noted that the truncated fluorescent protein does not emit any fluorescence, which rules out having transfected the original plasmid by mistake.
We checked the integrity of the primers by PCR and sequenced the source plasmids to see that they were not damaged. I verified that competent bacteria (DH5 alpha) were not the problem either.
Any idea?
Hello! So this seems like a job for serialcloner. I would put your expected sequence in the multiple cloning site of the plasmid and generate (or use) sequencing primers outside the MCS, which are usually suggested by the plasmid supplier, in many cases. Confirm with Primer-BLAST the gc content, length, and melting Tm match the requirements for sequencing. Then if you use IDT check for primer dimers or secondary structure with their computational tool. It is also possible to sequence a PCR product from most sequencing providers. Good luck!!
Matthew Edward Thornton Thank you for your response. I forgot to mention that the gene of interest coupled to the fluorescent protein is always located in the mcs. When sequencing we have tried different primers, some internal and some external to the mcs (e.g. T7 forward). We have also tried sequencing the PCR fragment, without success. The Tm of the primers and the %GC content and length have been verified and validated for sequencing. However, it may be interesting to analyze the possibility of having secondary structures or possible dimerization of primers, I will take a look at it!
Juan Mozas Perhaps a nick or linearize the plasmid. So in serial cloner which is free software it can estimate the fragment size from restriction enzyme mapping. In the olden days before sequencing they would do restriction mapping. Now it is generally a textbook exercise. In this case it could be useful. Especially if you have access to a number of restriction enzymes. Good luck!! Here is the URL http://serialbasics.free.fr/Serial_Cloner.html
Hi,
I think you got a lot of good tips from Matthew Edward Thornton however want I would like to add here is - double check your primer concentration and the requirements for the primer concentration for sequencing. To my knowledge the primer concentration for sequencing should be in µM range (1 µM = 1 pmol/µl) (as for PCR) not in the nM range.
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