ligase inhibitors - (Aug/18/2006 )
I'm trying to clone 1.8kb insert into 6.7kb vector. I digest the vector with AvrII and the insert with NheI and SpeI. After the digestions I get the right band pattern on the gel and I gel extract the insert using the qiagen gel extraction kit, I also remove the phosphates from the vector with Antarctic phosphatase from NEB which I subsequently heat inactivate.
I ligate with the rapid DNA ligation kit from Roche, and I get zero colonies. 
I also have a control ligation with the digested vector without the insert and I get zero colonies there as well.
I know transformation works because I have a positive control with the uncut vector and I get plates full of colonies there. So something must inhibiting the ligation.
Trying to figure out what's wrong I've tried to change several things in the cloning procedure. I've used different ligase (T4 DNA ligase from NEB), I' ve tried different vector:insert ratios, 1:1, 1:3. 1:5, I've tried with CIP instead of the antarctic, or not to remove the phosphates at all, I've used different cell lines for the transformation (DH5a cells from invitrogen, JM109 from promega).
When I run the ligation products on a gel I see a main band on the size of the vector, two higher molecular weight bands that I assume they correspond to circular and nicked plasmid with the insert and also a lot of lower molecular weight bands that are multiples of the insert size.
I believe the reagents that I'm using are fine because I don't have any problems in the other experiments that I'm doing, but I've had hard time to clone with this vector in the past too. I got only 2 or 3 colonies after several trials while it seems to work fine for other people that are using it.
Does anybody have an idea of what's wrong? What could inhibit the ligation reaction?
How old is ur ligase and ligase buffer?
How much of vector do u use for ligation? Use higher amounts of vector and different ratios for ligation, this can help as well.
I always say that its better to get even a single right colony than plenty of wrong ones.
So if u have 2-3 colonies with the right insert, b happy and enjoy the weekend.
When I run the ligation products on a gel I see a main band on the size of the vector, two higher molecular weight bands that I assume they correspond to circular and nicked plasmid with the insert and also a lot of lower molecular weight bands that are multiples of the insert size.
If you see that patron in your ligation DNA gel, I don’t think ligation is the problem.
Are you sure your cells are very competent? Did you calculate your efficiency?
How much uncut vector use as positive control? I used 0.05 ng of plasmid in my positive control.
I don’t know how much DNA you are using, but I would use more DNA.
The ligase and the buffer that I'm using are fresh.
I'm using 100ng of the vector and various amounts of insert, the maximum that I've used of the insert is 135 ng. Do you think I should go higher than that?
I never calculated the transformation efficiency of the cells but I'll do it, the truth is I using a lot of the uncut vector as positive control about 250ng.
Thank you for your ideas!
100 ng is a lot of DNA for a ligation, lower your amount of vector to maybe 20-60 ng, and vary the amount of insert.
Is your DNA the right quality (A260/280)?
What's your DNA dissolved in, Tris/water/TE, and what's the pH of the solution you've dissolved your DNA in?
Apart from this, are you heath-shocking or electroporating your bacteria (for electroporation there's too much salt in ligation reactions).
Had some bad luck with ligations recently as well (2 times I tried 6 ligations, had zero self-ligations, zero positive ligations and my cells were competent as checked with another uncut plasmid). I feared the problem might have been UV-damage to my DNA and I tried purifying without exposure to UV (SNAP UV-free kit from invitrogen, but you can also run a gel and have your vector or insert in 2 lanes, and only expose one lane to the UV, determine where your DNA should be in the other lane and excise then, put something under your gel on the part you don't want exposed). After this, I made sure I eluted my DNA in water (ligation is pH sensitive, so take care when adding large amounts of Tris to your ligation) and had succes from the first time" on all 6 ligations.
You are right, probably UV was damaging the DNA (or something during the gel extraction procedure). This time I used the vector directly after digesting it and the phosphatase reaction without running it on a gel and I got colonies. The problem is that I had colonies also in the negative control so I don't know yet if any of them has the insert in, I'm running the mini-preps right now.
I'll probably try this SNAP UV-free kit.
Thanks a lot!