We want to obtain a double stranded 120 bp long sequence and ligate it to a vector. The sequence is highly repetitive so we were not able to sequence it; therefore we were thinking of making it using sticky end ligation.
We will order complementary primers which will join to make double stranded short DNA (~45 bp) with sticky ends (6-7 bases). Then we want to ligate these sticky ends to each other and to the vector.
Has anyone tried this before? How should we go about setting up the reactions?
Thank you in advance!
If you are using Type IIs restriction sites in the vector the sticky ends generated on the vector will be unique and prevent self ligation of the vector if the cleaved 'stuffer' (original fragment in the vector) is removed (e.g. see fractionation in a gel). Your synthetic fragments can be arranged to have the complimentary 'sticky' ends to the linear vector fragment. A three-fold excess of insert fragments/vector (e.g. 6nMolar insert/ 2 nMolar vector) should work , but just to be sure try other concentrations.
Sara Mingu while it normally it is straightforward to order a few shorter oligos and ligate them to get your 120 fragment, if the sequence is really as repetitive as you suggest, then you are likely to have all sorts of cross hybridization problems. It might be easiest to order two single complementary full length oligos with restriction sites at the ends.
One problem you may have is that if you are truly unable to sequence across this region, how will you confirm that you have built the correct construct?
Dear John. I have retired since 10 years and had a stroke 5 years ago, I do not think I can give you a good answer. You are still at the lab bench? Best wishes! ErikL
- Badges
- Science method