I have transformed some DH5alpha cells with point mutated proteins placed in pcDNA plasmid and extracted the plasmid; now I have to send the plasmid for sequencing for confirming the presence of mutated proteins, and I am confused as to how to design the primers, first of all, I am unsure whether to sequence the whole plasmid which is ~6000bp long, or only sequence the region where I would have expected the point mutated protein to be inserted?
If I have to sequence the whole plasmid, would I simply have to design the forward primer right from position 1 of the plasmid, and the reverse primer to the reverse complement at the very final nucleotide of the plasmid and make the primers ~ 18-24bp long?
Thank you very much for your advice!
No, you wouldn't be expected to resequence the whole plasmid. Normally, you just sequence the full insert that you have introduced, just to check that it has the expected sequence, with the desired mutations if needed, and without spurious mutations that may have been introduced by errors in the PCR amplification. The main thing is to make sure that the proteins that you are going to purify and characterize are indeed those you designed. Of course, mutation of the vector can never be ruled out, and it could affect the expression level of the recombinant protein. However, it occurs rarely and if you take care of maintaining antibiotic selection for the presence of the plasmid and repression of the cloned gene until the time you need to express it, this is usually not a problem.
Dear Landan
As Pierre already told, in the most cases we are not checking the sequence of the full plasmid (expecially if you are using an high fidelity polimerase or you perfrom cloning with restriction enzimes) but the sequence of the insert and its flanking region. It is important check also the flanking regions (eg recombination site and 30-50 bp before and after the inser, just to be sure that there are not frameshift, mutation in promoter or lost of stop codons. In some cases, ag Topo cloning if you are not looking to the flanking region the insert may seems ok also if the insertion happen in the opposite direction (it is happen in a certain % of cases whith this kind of cloning)
Regarding primer desing, the number of primers and reaction sequence depends from the lenght of your insert, because generally with a single sanger sequencing reaction you can see a sequence from 500 to 1000 bp from the primer annealing point but you need other primers to see farway.
Therefore, If you insert is small (ag 1000bp) probably you can cover its entire sequence just with 2 primers a forward and a reverse annealing in the plasmid backbone, just before the insertion and after insertion of your GOI and in this way with only 2 common primers you can sequence many insert, but if the insert is big you have to design some additional primer that anneal into the insert.
This mean that if you wouldl like to sequence a full plasmid you need many primers.
Ciao
Manuele
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