I have fragment of less than 100 bp that I want to know the sequence of. Cloning will help with gaining a longer fragment but will not show the complete sequence. Any ideas?
Hi, I also worked with short fragments, so what I did was to put the fragments into a cloning vector such as pGEM-T (in case of Ts overhangs) or ZERO/TOPO (in case of blunt end fragments).
You then send your fragments to sequence using universal primers from the vector itself.
Hope it helps!
Sorry, this answer may be a little late. However, I did not see your question until today. For any interested, there is another way to sequence short fragments that worked well for us. We wanted to determine the sequence of a 100 base oligo to confirm correct synthesis. We used forward and reverse primers to amplify the product. The forward primer was 5' tailed with m13(-21). The reverse primer was 5' tailed with m13reverse. The first cycle in PCR generated the reverse strand to generate a double stranded PCR fragment. We were able to sequence the complete 100 base oligo using universal primers. It worked very well.
As Yulia Fridman said, you should clone your PCR product first then go for sequencing using universal primers. There is number of cloning kits are available, you can go with any one.
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