Ligation troubleshooting! Please help!
Posted 31 March 2013 - 05:16 PM
I am trying to clone a 0.6kb insert in 5.5kb pcDNA3.1/mychisA vector with restriction sites BamH1 and Nhe1. Sequential digestion recommended for this double digestion. Vector has treated with Alkaline phosphatase after restriction cut to avoid from self ligation. I ligated insert and vector with 3:1 ratio with T4 DNA ligase(its a newly bought ligase) overnight 16C. Ligation mixture transformed into dh5a Ecoli competent cell. Colonies were visible and I did colony PCR using forward and reverse primer which contains insert part (primers that i used to prepare insert). Colony PCR showed exactly same size band with my positive sample. I did dna miniprep from several colonies and double digested with the enzymes. But there were no inserts. I tried many times digesting with flanking sites and every possible sites that vector have. It was digested by BSTEII site which is only exists in the insert part. I suspect there might be an insert but I can not prove it. I sent my samples for sequencing. but the company said there was no reaction occurred. I really don't understand what to do for the next step. Is there anyone who encountered such a problem before? Please share and advice your ideas to me. I REALLY NEED HELP (
Posted 31 March 2013 - 05:45 PM
You should use one primer on the plasmid backbone, and another on the insert.
Your ligation is likely prevented by the alkaline phosphatase. I would definitely avoid its use. You should not need it with a double digestion.
The colonies you see are likely undigested or singly digested plasmid which transforms. I would try again with no AP.
If you continue to have difficulty, I would suggest using PCR for amplifying the plasmid, which will dramatically reduce background.
Posted 31 March 2013 - 10:25 PM
I have tried many times without AP. It was exactly same result as now also there were so many visible colonies. So i tried to reduce the self ligation possibility.
what do you think about it cut by enzyme that only exists in insert part.
Posted 01 April 2013 - 04:22 AM
if you cant cut out the insert using all possible cuttings from the vector then your colony PCR is more likely a false positive, so as your sequencing result (but make sure the sequencing failure is not due to the lack of purity).
About BSTEII digestion in the insert, did you run a vector alone digestion (BSTEII) along, to be sure it is not the star activity of the enzyme ?.
I always had an alternate hypothesis....
Posted 01 April 2013 - 09:25 AM
How was your BamHI/Nhel insert generated? Are you cutting out of another plasmid or did you add these sites to your primers and PCR amplify?
Edited by Ahrenhase, 01 April 2013 - 09:27 AM.
Posted 02 April 2013 - 01:35 AM
Ahrenhase, yes my insert contains restriction site and PCR amplified well.
I will try PCR and let you know the result, thanks for your precious advices.