ligation problem with SalI degested vector and insert - (Mar/29/2009 )

Dear all, I need your help! I am trying to ligase a salI digested pAC-GFP vector (clontech) with a DNA insert which is also treated with salI. Unfortunately, I failed every time. Here is my strategy. Could anybody help me check the problem if any. Thanks a lot!

Basically, I would like to cut a CAG promoter from pCAG-GFP vector and ligase this DNA segment into a promoter-less vector pAC-GFP. There are two cutting sites in pCAG-GFP vector, just in front and after the CAG promoter gene. So I cut the CAG promoter from pCAG-GFP vector using SalI digestion, ran gel and purified the DNA segment by gel purification kit. For pAC-GFP vector, I digested it with SalI for 3 hours at 37 degree and then treated with CIP for 1 hour for dephosphorylation. I tried the ligation ratio from 1:1 up to 1:7. I added 10ul of ligation mixture into 100ul of competent cells, or 4ul of ligation mixture into 50ul competent cells. I tried the incubation time as 4 hours at RT, or 16 hours at 16 degree. However, so far I could not get any result.

Does anybody encounter similar problem before? Could anybody give me some suggestions? Thank you very much!

I too have had some problems with SalI and cloning PCR products.
And I'm sorry, I wasn't able to have success with my project, so I'm not much use to you.
According to NEB, SalI has trouble cleaving PCR products -really wished my boss had looked it up before designing the whole cloning procedure around it!!.
Lucky for me, now that I am a student again, I could hand that project back to my boss to figure out.
If he does have any success (though he is not working on it at the moment) I will be sure to let you know.

Good luck

leelee on Mar 30 2009, 12:19 PM said:

Thank you, leelee! Seems I'd better re-design my procedure......

annie-fu on Mar 30 2009, 04:23 AM said:

Hello, sorry for a question that might seem obvious but when you say "I tried the incubation time as 4 hours at RT, or 16 hours at 16 degree. " I'm assuming you refer to the ligation, and then you transform, am I right?

Also, when you say "could not get any result", what exactly do you mean by this. No colonies? Colonies but no positive ones?

Do you have a positive control for your transformation? I think you are using too much DNA for your transformation, usually I use 1-2ul for 50ul of competent cells. What protocol are you using?

Re- SalI having trouble cutting PCR products, if I've understant your protocol properly, that shouldn't be your problem, as you are cutting your insert from a vector, arent you?

Leelee.... if you are having problems digesting a PCR product I'll recomend T-cloning firts, and then digest from this vector.

Hope this helps.

Since you are cutting 2 sites with the same enzyme, you may be getting self ligation of the vector and/or inserts (though CIP would help). I assume that you are gel purifying both vector and insert fragments prior to ligation, and heat inactivating samples after ligation, correct? If you haven't tried incubating RE and/or ligations reactions overnight, try it.
You may consider using different restiction enzymes. Find out which REs are present in the vector that you want to insert the promoter into, and design custom primers (with RE sites added on) flanking the promoter region, then PCR to amplify the promoter and thereby adding on the new RE sites. RE vector and insert and continue with ligation.

almost a doctor on Mar 30 2009, 07:48 PM said:

Hello. For your first question, yes I did the transformation after ligation, but no colonies appeared after overnight incubation. However, for the positive control, the protocol worked properly. Maybe I need to reduce the DNA amount...

Thank you so much for your suggestion!

NKatpase on Mar 31 2009, 07:07 AM said:

Yes I did the same procedure as you described, and I also tried digestion and ligation overnight, but no colonies appeared. My boss assumed the quality of the ligase enzyme was not good and asked me to order another one. I will update you if my problem solved. I will also try PCR simultaneously.

Thanks a lot!