Hi all. I designed a plasmid construct inserting a gene about 5.5kb long. After sequencing, I am getting a 18 base deletion around the 3 prime region of the construct. I have tried it many times but every time I get this 18 base deletion. Should I redesign the sequence again? If so, what should be kept in mind to get the whole sequence intact. Thank you all.
you should design your primer such that the 5' end starts from Restriction site and ends on restriction site; because sequencers have a maximum capicity of 600bp in the end some base pares might not be readable. try with both reverse and forward primers to sequence. also keep in mind Prashar suggestion.
Good luck
Did you design both forward & reverse sequencing primers? As Qari said, sequencers have a limit (i.e it can not sequence the DNA longer than certain size). Make sure to design reverse primer at least 100 bp downstream of 3' region. Also, check if the enzymes you're using cuts inside the gene of your interest. It should only cut before 5' and after 3' ends, not in the gene. Good luck ;)
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