Nde1-BamH1 makes me sad - high background despite DIFFERENT methods (Mar/10/2010 )

prehaps the overnight digestion is the problem. Most restriction enzymes i use such as BamH1 only require a 2 hour incubation period at 37 degrees to digest samples efficiently. that could then reduce your background troubles

and for ligations i use T4 DNA ligase (NEB) and find it works best when you leave your sample at room temp for 2 hours

Hi everyone, I am here to update on what I've done so far.

I started off with a fresh miniprep of pet22b vector, out of which I took 20ul for double digestion with Nde1 and BamH1(HF) since they are compatible in NEB4. Instead of digesting overnight, I did the digestion at 37degC for 2 hours. The reactions were cleaned up using a PCR cleanup kit since gel purification has been said to be able to inhibit ligation. A little of the cleanup was then used for a test ligation (just the cut vector alone without any inserts) with T4 DNA ligase.

I then took 50ng of the fresh miniprep, 50ng of the cleaned up double-digested pet22b vector and 50ng of the test ligation for transformation with HIT cells. I got a TONNE of colonies for the fresh miniprep, which was to be expected. The cleaned up double-digested pet22b vector had a number of colonies so a small fraction of the vector was uncut, which was fine with me. But the test ligation had the colonies filled up the plate, which was really depressing. This would show that single cut has been happening.

Phage434, I was bringing up your point of ligating single cut vectors to see if double fragments would appear. But, the effective concentration of the two compatible ends on the same linear single cut fragment is higher than two ends on two linear single cut fragments, isnt it? As a result, a majority of it would self-ligate instead of inter-ligate and we would get a circular product, which is the original fragment itself.

On one hand, I am wondering if I should move on to doing sequential digestion to see which enzyme is not cutting properly, but this would just waste even more time.
And Dukey and Denny... I think I am most probably moving on to finding another restriction site to go with Nde1. This has been wasting a lot of my time. As I am doing cloning of a mutated library, I will need double digested vector backbones.

I thank everyone for your ideas, suggestions and advices. They have definitely helped a lot and I will incorporate them in my work.

wizzkid on Mar 12 2010, 11:40 PM said:

Hi wizzkid,

I did that. The ligation is working well. The problem lies with the digestion. =(

jiajia1987 on Mar 15 2010, 07:23 PM said:

The problem almost ALWAYS lies with the digestion, most specifically the second digest as this one you cannot monitor on the gel. That is where you need to focus your attention.

just a quick question, how far apart are the NdeI and BamHI sites? NdeI requires 8bp skirting the site for any efficiency at cutting.

perneseblue on Mar 19 2010, 08:14 AM said:

Hi pernesblue,

They are about 80+bp apart on the pet22b(+) vector.

Dukey on Mar 19 2010, 01:50 AM said:

Hi Dukey,

Yes the problem lies with the digestion. A postdoc from my lab helped me to run a gel. I did sequential digestions with either enzymes eventually. Nde1 seems to be causing a problem as it has a very faint band corresponding to the main band that the undigested vector gave. BamH1(HF), on the other hand, gave a clean band.