Posted 07 June 2013 - 04:23 AM
I am trying since some time to ligate adaptors to blunt ended sequences, but so far no success.
I am using DNAse1 to randomly cut my sequence of interest and T4 DNA polymerase to fill the ends. Then I add two annealed adaptors, one is 5'AAACCTCTCCGGAGCCTGA 3' and /5'Phos/TCAGGCTCCGGAGAGGT 3' for the 5' end and 5'GAGGTGTCGCCGGTCA 3' and /5Phos/TGACCGGCGACACCTCTCCGGAAAA for the 3' end and ligate O/N at 16C. Next I wanted to do a PCR using the unphosphorylated adaptors as primer, but no product.
I have also tried to use Taq polymerase with random primers to add an A overhang to the digested sequence and ligation with single T containing adaptors as well as blunting and ligation of didesoxyTTP together with an A overhang, but nothing. The ligase works well with all other stuff.
Any idea what to improve? I hope it's a stupid mistake, like adaptor sequence or too high concentration of DNA...
Thanks a lot,
Posted 08 June 2013 - 01:59 AM
Posted 10 June 2013 - 06:53 AM
Do you think that conditons of adaptor annealing are important? I usually mix 1ul of 100uM for/rev adaptor in water, 95C for 3mins and cool to RT with 0.5C/sec. Most people use some buffer (1x ligase buffer) and a slower cooling rate...
Posted 10 June 2013 - 12:31 PM
Posted 13 June 2013 - 04:56 AM
Sorry, I am a bit desperate Thanks for your help.
Posted 13 June 2013 - 06:21 AM
Posted 13 June 2013 - 07:40 AM
My goal is to integrate a defined stretch of amino acids into a target peptide at a random position (insertional mutagenesis). The target DNA is ~750bp, circular. The nucleotides are 27nt long, and I'm struggling with it since almost a year Fortunately I have other projects as well. I would be very happy if anyone can suggest something.
Posted 13 June 2013 - 12:19 PM
I suspect that a combination of blunt end polishing, followed by A tailing, followed by ligation would work. Do you have a way of selecting for success? How will you know you have the correct result?
Posted 14 June 2013 - 04:48 AM
My selection would be a PCR with AAACCTCTCCGGAGCCTGA and GAGGTGTCGCCGGTCA, which are part of the original adaptor duplex. Do you think I can get a PCR product? Maybe this is the problem that my detection method doesn't work...
Posted 14 June 2013 - 05:00 AM