Do I have to purify vector after RE digestion for cloning? - (Jul/25/2006 )
Do you worry about possibility of mutation by this method?
if you can't get your vector to religate as a positive control, there are a number of possibilities, all of which can be tested in turn:
1. your ligase is bad
2. the ATP in your ligase buffer has croaked (I would check this first!)
3. something in your DNA purification is a) degrading your DNA or 
inhibiting the ligation
do you ever run an aliquot of your purified vector to be sure you have good yield and intact DNA?
have you tried new buffers for everything? (a dirty batch of tris can mess up a lot of steps 
)
have you tried new ligase?
1. your ligase is bad
2. the ATP in your ligase buffer has croaked (I would check this first!)
3. something in your DNA purification is a) degrading your DNA or
inhibiting the ligationdo you ever run an aliquot of your purified vector to be sure you have good yield and intact DNA?
have you tried new buffers for everything? (a dirty batch of tris can mess up a lot of steps
)have you tried new ligase?
I have tried 3 ligases now...and my labmate has used 2 of them successfully....definately could be off ATP...how does this happen??
Do mean something in the plasmid prep or the gel purification?? I do run out my pure vector and spec it...it is all good to my knowledge....no smearing or anything.
I am starting to really look at the buffers I guess and the possibility of damage during gel purification.
So you say..stain quick with EtBr and rinse or stain with something else....is it CYBR green or something??
Cheers....nice name by the way
Ames-
thanks
the ATP in ligase buffer is notoriously unstable; if you don't use up the buffer relatively quickly then it's a good idea to get the buffer and the ATP separately and make fresh aliquots from time to time (or just order extra and throw the tubes out periodically). as with everything in our lives, avoidance of unnecessary freeze/thaws is always good
when I say, do you run the DNA out, I don't just mean of your vector...after you cut, run, and purify your fragments, just prior to ligation, take a little and run it again to be sure it's all there and it looks intact (also gives you an idea of how much you've lost during purification)
normally, this is not necessarily necessary...but if you are having troubles it's an excellent control
for your purification...limit EtBR, limit UV for sure! and try a few different methods, even if you have a ton of DNA I always use the 'low yield' modifications...for ex, heated elution buffer, that sort of thing...do you see what I mean? you lose so much every time you purify
My points:
1) Definitely sub-aliquot Ligase buffer immediately after the first thawing of stock. I like 50 ul.
2) I would advise purification after restriction digestion on a gel of the vector backbone. It is quite frequent that a large portion of the vector backbone will remain uncut or digested only by one enzyme. Purification will remove the undigested backbone.
3) Always perform gel purification with low-melt agarose. Regular agarose has enzymatic inhibitors and doesn't liquify as readily as low-melt, thus drastically decreasing the efficiency of ligation.
-Matt
I would not recommend pcr amplification of the vector backbone because it is cumbersome and expensive. Gel purification is the ideal and practical alternative.
-Matt
WOW thanks....so much to work with...I will let you know how it goes
Cheers 
Well it looks like it might have been the cells after all....tranformable but really low efficiency. However, to ensure problems I am taking care of all these little things now .....thank you 