Ligation troubleshooting! Please help! - (Mar/31/2013 )
Hello everyone, I am suffering with ligation work for several months. I really need help!
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 (
Colony pcr with primers that bind only to the insert is a bad strategy. The insert DNA will still be present on the plate, and will amplify it.
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.
Thank you for replying,
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.
Are you sure both the Enzymes worked?? how much vector used for digestion? may be if you used too much (less time) your digestion wnt be efficient, and that ll give you background, alternatively you can PCR as Phage suggested.
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'm having a similar problem with non-complementary double digest.
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?
Actually I am really confused with this now. I am not sure that was star activity or not. and I used kit for dna isolation.
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.