Ligation optimization - (May/16/2005 )

Hi!

For the past few weeks, I have been having problems with getting colonies on my plates after ligations. I generally get 1 or 2 colonies if I am lucky, and most of the time, the colony does contain my insert but I would like to increase the number of colonies I get.

I have tried a couple of different ligases and followed the protocol exactly given by the manufacturer (3:1 insert to vector molar ratio), but still no luck.

I have tried:

Quick ligase from NEB
T4 Ligase from Invitrogen
T4 ligase from NEB

During transformation, I pellet all my cells so I can plate all of them.
My competent cells are freshly prepared and are good (I have already done a positive and negative control on them).

Any suggestions or advice would be appreciated!

Thanks!

RV

Ligations suck ... that's the long and short of it. It depends on your experiment (ex. sticky vs blunt). You can increase efficiency by killing the ligase (ex heat denature) prior to transform. You can improve transformation with reducing agents (B-mercEtOH, DMSO).

A lot of good stuff can be found here:
http://web.mit.edu/bevin/www/Genejock/Gene...ng%20-%2002.PPT

did you cut with one or two enzymes? Are you certain that you've cut to completion?

I've found that ligation always works for me. It's a matter of preparing the insert and vector properly.

The only time I've had trouble obtaining a number of colonies is when transforming with large vectors (18kb and up).

I cut 1 ug of DNA with 20 units of one enzyme (BamHI) for 2 hours at 37 deg celsius and I dephosphorylated the vector using CIP. Then, I ran both insert and vector on a gel to make sure its been cut to completion, extracted the bands from the gel using the qiagen gel extraction kit.

* limit the UV exposure of your DNA, especially with short wave UV. If possible, use 365 nm long wave UV to visualize your gels

* limit the amount and time of action of your CIP. Use only the number of calculated units, not an excess.

* Heat anneal the vector/insert mix prior to adding ligase, if doing sticky ended ligations (60 C, cool slowly).

* Allow the cells to recover and grow for an hour in antibiotic free medium prior to plating out (not important for Amp selection, but important for e.g. Kan)