Dear all
Please if someone can guide me.. I have cloned a gene of 1500 bp pairs in FAST-Plasmid. I used reverse primers to sequence the gene. The company sent me 900bp length.. I want to sequence the remaining portion of the gene... Please if someone can suggest me how to get full length sequence of the whole gene from company...
Hi Usman,
You'll need to sequence with a forward primer, and design some internal forward and reverse primers to obtain full length sequence of your gene. I assume you are using Sanger sequencing, which has a maximum read length of approximately 700-750bp before the base call signals start to deteriorate in strength. So for a gene that is 1500 bp in length, you will need at least two sets of primers (forward and reverse at either end). If you do not get any overlap of your sequences from two sets of primers, then you should design one or two more internal primers so that you will have at least one overlap, or 2x coverage, which is the minimum requirement for confidence in sequence of a contig (sequences that assemble into a contiguous sequence). Hope this helps.
Cheers
Shan
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