I am out of my wits trying to get a ligation reaction to work. It would be very helpful if you could give some advise. The following are the conditions of the ligation reaction I am trying.
1) PCR amplification of template DNA (~3kb size) and gel purification using QIAquick purification kit.
2) Restriction digestion of pLenti6/V5 TOPO vector (~7kb size) with BamH1 and BstB1 for 1 hr at 37C. Gel purification of vector and hopefuly I did not overexpose the vector DNA to UV light while cutting the band from the gel.
3) Digested amplified template DNA with BAMH1 and BstB1 for 1 hr at 37C. Gel purified digested DNA with, what I think was, very little exposure to UV light.
3) Treated vector DNA with Antarctic phosphatase for 60 mins at 37C and then denatured enzyme at 65C for 8 mins.
4) Ligation reactions:
a) tried to ligate 58 ng vector + 75 ng template at 16C overnight
b) tried to ligate 58 ng vector + 75 ng template at 4C overnight
c) tried to ligate 58 ng vector + 100 ng template at 4C overnight
I did ligation reactions with all the above conditions to just test the ligation reactions on a gel to verify and saw a band at 10 kb and higher for ligation reactions and just vector and template bands in a control reaction without ligase.
I tried to transform stbl3 bacteria with the ligation reactions and I do not see any colonies. My positive control with circular vector plasmid was able to transform the bacteria but none of the ligated plasmids.
I am at my wits end trying to figure out what I am doing wrong.
Any advice will be be helpful at this stage.