I am planning to perform gene deletion using pKOV vector method. However I am facing problems in the initial ligation step!! I have amplified the fragment of interest (upstream and downstream regions of the gene to be deleted) and by overlap pcr both fragments were joined together. And I have purified the overlapped regions after pcr by gel purification kit (favorgen). The pcr amplification were good and no problems were faced here. Also the pkov vector is also in correct size (5.6 kb) after double digestion with BamHI and NotI one after another. Similarly i have digested the insert with BamHI and NotI enzymes one after another. After every digestion of vector and insert they were gel purified and I have set ligation at 16C for 16hours. After transformation, I cant even get a single colony out of it. On transforming the vector alone I got many colonies which confirms the quality of competent cells I am using. I have repeated this several times but so still I am failing to clone it. even tried it with different ligation ratios!! Simultaneous to the start of this cloning work (5 months back) I was able to successfully clone two different types of insert in pCDF vector, only with this vector I am facing problems in the cloning. Recently I suspected that whether ligation is affected due to gel purification step? because during two succesful cloning in pcdf vector due to very small release size I didnt go for gel purification, however on doing a new cloning attempt with another insert, I went for gel purifiaction after digestion. Surprisingly I didnt get any colonies after transformation, and on planning to further proceed with optimizing the ligation ratio, I thought of this gel purification problem caused by UV exposure. Approximately during purification, the gel could have been exposed to UV for 30 to 60 seconds! SO can anyone check out my issue and tell whether UV exposure during purification is the only mistake I did? If so suggest some remedies to overcome this. If not are there any other problems in my cloning step??
Edited by sathya05, 18 April 2022 - 12:19 AM.