It seems to me that you have a problem in the restriction enzymes. and concerning false negative its natural mutation due to the heat shock which is common sense in cloning

If I had a problem in restriction digest wouldn't I need to get band when I perform PCR with purified vector after digestion. Ones my vector has been digested primers can not amplyfy the region. And I got no band in this PCR but control reaction with non-digested vector gives the exact band.

You could check the following:

1- what resistant gene does the vector has? Check the plasmid map on add gene. Your plasmid may contain Amp gene and not Kanamycin.

2-Did you put the restriction sites in your primers on the 5' ends?

3-The buffer used for the double digest, is it compartible for the two restriction enzymes? Check compatibility on Neb biolab.

4-What ratio of vector/insert did you used? 1:3 is good. To calculate your vector/insert ratio, check Neb calculator, choose ligation and then put the corresponding vector and insert size and concerntrations.

5- Check if the two restriction sites have thesame overhangs (just like MluI and BssHII having thesame overhang). If they do it could be the reason for the self ligation. What you can do to prevent self ligation is to CIP (dephosphorylate) the vector using Alkaline phosphatase. (check protocol on neb biolab. Note! Dont CIP the insert.

Good luck!

@ Elvis

Dephosphorylating was crucial to obtain a good plasmid in my last project, this should be standard. Definetely worth a try.

@ Enes

Depending on your insert size compared to vector ratio, just add a bit more insert (I used the calculator, and then doubled the insert amount). Try fresh enzymes, solutions, etc

A theory raised by my supervisor was that while plating out. the unligated insert (still in the mix) could be taken up by cells even though it wasn't correctly ligated (a big insert could then self-ligate, basically creating 2 vectors in one bacteria). So plate positive colonies on a new plate to confirm. Also, you can do a colony PCR with primers binding inside and outside the insert.

Also make sure you don't add antibiotic to (too) hot media. It might be simple and standard procedures, but after a lot of plate pouring you might not notice the real warmth of your bottle (but your NC are fine, so this should not be the case).

Thanks for all answer;

@elvis,

I have kanR. I am using Sgfi and mlui. I have added 5 extra 5' nucleotides to primers. I perform experiment with 1:3 1:5 1:10 and 1:20 ratios.

@ruud,

My enzymes are working well, I have checked them in a different experiment and they work fine. my colony PCRs are always negative that is the reason I asked why I get these colonies after 24h. I add antibiotic at enough cold temperature.If my antibiotic was the problem I could get colonies earlier.

Maybe you can design new primers starting around 50 bps in the vector backbone and reading into your insert and do sequencing to confirm the presence of your insert.

Also you can use Blue/white screening if your vector allows to confirm positives from negative colonies.

Maybe try to do a restriction digestion analysis using restriction enzymes other than you use for insertion to confirm the presence of our insert.

Good luck with your cloning.

Need better insert to vector ratio. It’s re-ligating without insert. So add more insert per amount of vector