Ligation Problems - (Jan/09/2010 )
I've been trying to ligate a 1.1kb fragment into a 9kb backbone. The protocol I've used so far, consists of linearizing the vector with NEB SalI-HF. The 1.1kb fragment was amplified by PCR, purified using a QIAGEN PCR purification kit, and digested with NEB SalI-HF.
After digestion of the backbone, it was treated with Invitrogen calf intestinal alkaline phosphatase, but putting 1U of it into the 20uL digestion mixture, and incubated at 37C for 5 mins. The CIP was then inactivated by phenol-chloroform method.
I took the two pieces to be put together, and ran them on a gel along to verify the cutting. Both showed as sharp distinct bands. They were also run with a high mass ladder to approximate the concentration of the fragments.
Following this, they were put into a 10uL ligation mixture using NEB T4 DNA ligase, at a molar ratio of 3:1. Ligation took place for 1 hour at RT. I used another ligation reaction containing no insert as a control as well. 2uL of each reaction mixture was electroporated into competent cells.
After overnight incubation, I'm still seeing about 30-40 colonies from both transformations. I've tried transforming undigested backbone, and am getting essentially confluent plates. I've tried screening the colonies by miniprep/PCR, and am still not seeing any clones that retained the insert.
I've been at this for a few months, and consistently, it looks like the insert isn't being ligated in. Furthermore, I've tried multiple ratios, 1:1, 3:1, 6:1, and 10:1, and none of these seem to work. None of the reagents I've been using are expired, so I'm really unsure of the problem. If anyone has any suggestions or advice, it would be greatly appreciated!
If at all possible, switch to a different enzyme. SalI is a known troublemaker with PCR digestions. Do you have sufficient 5' overhangs on your PCR product? Is there a different site in your backbone vector that you can use? If not, consider PCR of your backbone vector to insert a different cut site (two different cut sites, ideally) and change your PCR primers for your insert correspondingly. This will largely solve both your background problem and your digestion problem.
1 h for a ligation reaction might be to short ...i think NEB suggest doing a stick end ligation at RT for 2 h ...maybe you can try ligating longer, e.g. over-night @ 16°C or 4°C.
You added some extra base pairs with your SalI restriction site on your PCR-product? You have to add at least 4bp for SalI to work on your product (for detals watch this).
You can also try to do the vector only control with and without ligase ...so you will see if the background results from re-ligation due to incomplete CIP treatment or contamination of uncut vector.
Good luck for your further experiments!