Try to use 1:5 vector to insert ratio(molar). Also I would recommend using more vectors : at least 50 ng and take the annealed oligos accordingly. Check your ligase and ligase buffer; are they working for some other cloning? Avoid freeze-thaw of ligase buffer, try to keep them in small aliquots.

I tried differents ratio, and iI check my ligase and ligase buffer (I ordred them 2 weeks ago). And i kept them in small aliquots. But i think that the problem comes from the gel purification! what do you think about?

Did you check the gel purified product by running it on agarose gel? What method are you using to purify? If you use column based method I would recommend elute using the supplied elution buffer and keep the elution volume less ( about 20 ul) to get concentrated purified product.

hey our lab manager has had that problem for months. She had been doing overnight digests, and after ligation, she would have no colonies. She finally did the digest for 2 hrs and subsequently the ligation worked. She only had 2-3 colonies....but it worked and now she has moved on to the next phase of CRISPr.

@Samala Lewis

As you can see from the protocol given (...In total of 100 µl, 2 hours at 37°C...) Amine Bouchareb is doing a 2 hrs digest!

This is my product, on left the maxiprep product and on right the gel purified product after BsaI digestion

I've never worked with that vector but I've worked with virus vectors a lot and under my experienced, sometimes the ligation works better if you dephosphorylate the vector and phosphorylate the insert. Also in different virus vector I've only been able to clone using low melting agarose ligation. Good luck.

My insert is phosphorylated but for my vector it's not important to phosphorylate it.

i agree with you, some times agarose can inhibit ligation.

Yeah, I know. Usually when the extremes are cohesive it's not necessary to phosphorylate the insert and dephosphorylate the vector but sometimes the ligation works better.

Also if you want to try the low melting agarose ligation just let me know and I could give you the procedure that I use.

Yes I want of course, it will be interesting to try the low melting agarose ligation. Thank you very much.

Here is my procedure.

1. Cut 2-3ug of vector. 2-3hr is enough.

2. Cut 2-3ug of insert. If it is PCR product, I will cut overnight.

3. Clean the reaction with any commercial kit (I use quiagen and resuspend into ~40 ul)

4. Dephosphorylate the vector and phosphorylate the insert ( I use CIP and PNK)

5. Prepare low melting gel (1% in TAE and add EtBr, do NOT boil in microwave)

6. Load ~20ul of vector and ~20ul of insert on low melting gel. Run enough to get good separation.

7. Cut out the bands carefully and transfer to tubes. Gel is very soft. Minimize the size of the gel. Usually I got ~ 20ul after cutting.

8. Heat the tube at 70C for 5 min to dissolve gel. Cool down in 37C for 1 min before you add gel to ligation mix:

Vector (in gel) 2.5ul

insert (in gel) 6.5ul

10X ligation buffer 2ul

ligase 1ul

DW 8ul

Mix well (up & down) and incubate at least 3hr or overnight at room temperature or 16C.

9. Here you have two options:

a. Heat the tube for 2-3 min at 70C and use 1-2ul for transformation (Not always works for me)

b. Heat the tube for 2-3 min at 70C and clean the ligation with any kit that allows you elute in a small volume (10-12 ul) and use the whole elution for transformation.

Good luck!

Thank you very much. So "to be continued"