OK - there are two possible issues here, the ligations and the Blp1 digestion.

Blp1 and EcoR1 require buffer conditions that are not compatible. So by adding R1 to your Blp1 digestion mix, chances are you're not seeing much if any R1 digestion. That may likely feed directly forward to your apparent ligation problem. Why "apparent"? Because you may not have any fragments to ligate in the first place.

How are you checking the efficiency of your digestion? ARE you checking it…? Are these the only two enzymes you can use? Have you considered PCR-ing the insert out of wherever it is with "better" ends? Finally - you say you did get small numbers of colonies - did you look to see if they contained the clones you'd like?

It is easy to check your ligation technique and the issues around ligation are well known (dephosphorylation, for example).

Good Luck

G

Hi Anar!

I'll try to help if I can with possible things/tips for each step.

First a silly question: After CIPing, are you doing anything to remove the CIP before ligating your vector? If not, it may be hanging around and attacking your insert. I'm not sure why you are CIPing if your vector is also digested with EcoRI and BlpI since those don't look to give compatible overhangs. I use NEB's Antarctic Phosphatase (Cat #M0289S), which is heat-inactivated and has always worked great for me. If you are removing the CIP, here are other things that may help.

*** Digest ***

If you are using enzymes from NEB, their EcoRI-HF can help with the digest part (as opposed to the regular EcoRI) as it can cut in the same buffer as the BlpI.

If you are having a problem with low yield I would do a digest as such (assuming NEB enzymes):

TO GET THE GOI FROM pGBT9:

3-10ug plasmid

4uL 10x Buffer (depends on which EcoRI you're using

Water to 38uL

- if doing sequential, add 1uL of the first enzyme, mix, and incubate 1 hour at 37C, column purify and digest as above with the second enzyme

OR

- if doing a double digest with EcoRI-HF, add 1uL of each enzyme, mix, and incubate 1 hour at 37C

DIGEST FOR DESTINATION VECTOR:

For destination vectors I generally digest no more than 1ug in 40uL using 1uL of each enzyme. (Again, you shouldn't need to CIP your vector if you are cutting with two enzymes that will give incompatible overhangs so you may be able to cut this part out altogether.)

Run on a TAE agarose gel (I do 30 minutes at ~150-170V) and cut out and gel purify the bands you need. Unless your GOI is very tiny, you should have plenty of DNA for ligations after doing a gel purification.

*** Ligation ***

For the ligation, I generally try for a 1:3 or 1:6 molar ratio of vector to insert. I use NEB's 5x ligation buffer for a 15 minute ligation in 10uL total reaction then dilute it 1:10 using 90uL milliQ water and electroporate 1-2uL in 40uL electrocompetent cells (we make our own that have a 10^9 efficiency).

- 25-50ng Vector

- 3 or 6-fold molar excess insert (or same volume water for - control)

- 2uL NEB 5x Rapid ligation buffer (Cat #B6058S)

- water to 10uL

- 0.5uL T4 Ligase (I use Promega T4. It's cheaper and works great)

Hope this helps! Any other questions, let me know. I won't try to be as wordy next time =)

Thank you a lot Grant and Jean.

1. to Grant's questions: Yes. I've checked the digestions by low-melt and I got the predicted bands, which i excised and used for ligation. My concern is that even though Blp1 cuts, it just leaves weird ends and maybe show some exonuclease activity and the ends are just not ligating; I am also checking if i have the right colonies (the few ones i have) today.

2. to Jean: yes, using CIP was not necessary. And CIP is not heat-deactivated. But, it should not be a huge problem, since i do my ligations overnight at 16-18C. in terms of the buffer for the enzymes, since i got the correct size bands, i believe both enzymes are working in EcoRI buffer. However, the ligation method you're using seem to save much more time rather than incubating overnight as we do. For that, is the efficiency of ligation high? 15 mins is enough?

Hi again, Anar!

Yes, the efficiency of the ligation is very nice. It requires the use of that buffer, however (or any rapid ligation buffer) and should not be done with regular, non-rapid ligation buffer (especially if you are having problems with efficiency). The actual protocol allows for 5-15 minutes at room temperature but I usually do 15 just because it gives me time to prepare the cuvettes for electroporation =P

If the CIP is not necessary, I would avoid using it. It won't help and it could hurt. I'm not sure what CIP's activity is at 16C but I'd lay odds that it isn't 0%, which means you could be dephosphorylating your insert and drastically decreasing your ligation efficiency.

I'd suggest first trying to digest enough insert out that you have a respectable reading on your nanodrop (or other spec) and then trying the overnight ligation again with no CIP. If that doesn't work (or even if it does), try the rapid ligation buffer.

According to my experience in cloning, the most important factor in purity and concentration of your digested products before preparing ligation reaction.

I would consider that the digested ends are compatible and you choose the digestive enzymes according to the sequence and there is no problem at this stage. Then I would suggest to use nuclease free water to dilute the digested insert and vector post gel extraction. AND then concentrate the diluted product to make sure you have high quality and quantity products to start ligation reaction. Use 1:3 ratio for cohesive ends and 1:5 for blunt ends. Overnight at room temperature in 1 10ul reaction, and transform 2-5 ul to your competent cells.

hi

check your restriction enzyme buffer for cutting efficiency. do restriction reaction at least 10 hours if don't using fast digest enzyme. inactive restriction enzyme and purify restriction product then use insert ratio 3.1(attached) and put ligation reaction at degree of centigrade for 10 hours or 8 degree of centigrade for at least 6 hours. use newly bought t4 enzyme buffer (ATP has sensitivity to ice.thaw) or add ATP to your reaction. finally mix all of ligation product to newly prepared competent cell that placed at least for 3-5 hours at refrigerator.