pKOV ligation - (Apr/18/2022 )

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??

Without exact protocols it is hard to say what might have gone wrong. We need at least the results from your experimental controls (ligation, transformation, digestion).

What are the results of your controls? Do you get any background on your "no ligation" control? How about on your transformation controls?

What molar ratios of insert:vector did you use?

There are a few things you can do in the mean-time

First off, make sure your transformation is working - use another vector (many kit competent cells come with a pUC variant for this purpose). Also transform the uncut backbone that you are using. Determine the transformation efficiency of your cells and ensure that your antibiotic selection is working correctly.

Is your ligation is working properly? You can do this with an insert that you know works in a backbone that you know works. You could also test to see if you can ligate the bit of the backbone you cut out back into your vector.

Check your primers - did you add 6 bases 5' of the restriction site to ensure the REs can cut properly. If not, did you order phosphorylated primers?

30-60s of UV is very long, especially if you are using short-wave UV (most imaging platforms are, long-wave UV is relatively rare). You need to keep this down to <10s. You can do this by marking the band rather than full cutting and brief exposures to ensure you got the right bit marked. Cut fully, remove band then expose the gel (without the band) again to see if you missed cutting out the band. If you can't see the band on the gel without taking a picture/long exposure, you won't get enough out of the gel extraction to do anything with.  You need at least 1 ug of digested DNA loaded onto the gel in as small a well as possible. Thin wells (<1 mm width) work best.