Serious cloning issues - gel extractions?

[JackBean]

I think you can inactivate the AP also by heat or phenol:chloroform, check your supplier's web page That might help

[JackBean]

But I wouldn't recommend to mix it for ligation with active AP, as it will dephosphorylate ATP

[TaintedCherub]

I didn't know that! Thanks I will phenol:chloroform extract to be on the safe side.

[JackBean]

I by myself don't like phenol:chloroform. Too much work, smell and not sure recovery
I prefer the termo inactivation

[TaintedCherub]

I by myself don't like phenol:chloroform. Too much work, smell and not sure recovery
I prefer the termo inactivation

Will inactivated BamHI and CIAP be a problem for the ligation reaction?

I seem to be recovering a good amount from the phenol:chloroform extractions. My advisor showed me some tricks to minimise losses). Agree about the smell and work though

The latest thing I have done is to run a gel with the supplier's original vector straight from the tube and my preps.

I am seeing an extra band at about 550 base pairs in my preps that isn't present in the original vector. Does anyone have any idea what this band could be and whether it could be the reason for my cloning problems?

Gel: http://crayfish.zenfolio.com/p743268062 ... 9#h2eee5e9

I am doing a fresh vector prep from the original tube just in case. Be nice to know what the band is though. Prepped from XL-1 blue cells.

[JackBean]

If you purify it through phenol:chloroform, you shouldn't have there any proteins

I don't understand much the last thing with the band of 550 bp

[MrMistery]

your lane 3 also seems to have another band at ~1000. I honestly don't know what's going on, but it can't be good. I would just try it again with fresh vector

[TaintedCherub]

No, can't be helping.

I did some fresh vector preps, and still seem to have too many bands... I could justify three bands by supercoiled, linear and circular, but I have four. At least they're in different places. Maybe this is progress?

The six preps come from three single colonies. Image below.

http://crayfish.zenfolio.com/p743268062 ... #h22b69119

[MrMistery]

maybe what you're seeing is just a mix of topoisomers (in your new preps). Just cut it with a single enzyme that has one cut site - if you just get a single higher band, then that's what was happening. Now, why you would have more than one topoisomer, I do not know

[TaintedCherub]

I have done that. I got a single band, looks good.

Then I gel extracted and ended up with no DNA.

Anyway, doesn't matter; I had a meeting with my supervisor, we've shifted priorities and I will have to abandon this. Pity as I think I was getting close, but I really don't have time to be messing about any more. I will get in touch with a company (www.geneart.com) for a quote on farming the whole thing out.

Thanks for your help!

[priyajoshi]

For all of you newbies to the cloning process, may I suggest a free software tool to eliminate restriction mistakes? BxSeqTools is a very interactive site with a virtual cloning construct tool (look in advanced tools). There is also an "ultimate cloning guide" with some newer techniques given also.

http://www.bioinfoman.com

[chauj]

I have tried to simply ligate BamHI digested, gel extracted linear vector back to itself. 100 ng DNA, 1 U ligase for 10, 60 and 120 minutes at 37oC, using fresh ligase. None of these reactions resulted in any colonies, despite uncut vector producing a near-lawn. I have been using an estimated 10 ng DNA in transformations.

The only hint I have is that, following gel extraction and precipitation, there is always a peak in DNA absorbance at 220-230 nm, resulting in 260/230 ratios sometimes below 0.5 (260/280 is usually 1.6-1.. Also, during the latest precipitation, I noticed white flakes forming that were insoluble in water, which I span down and removed.

It seems like something is carrying over from the gel extraction that is either inhibiting ligase or killing my supercompetent cells. Possibly guanidium salts or agarose contaminants? I have now tried both Qiagen and Invitrogen gel extraction kits (silica column types) with identical results.

Does anyone have any experience with similar problems?

Might be a little late in reply, but hopefully this helps others that have similar issues:

I had cloning issues that sounds very similar to yours (somewhere within the gel extraction step). I finally found the issue was related to the quality of the agarose. I tried 4 different brands of agarose (normal agarose and low melting point agarose, old and new), each yielded DNA. However, transformation with 3 different brands of agarose did not yield any colonies (gel extracted uncut vector). Contaminants in old agarose or how the agarose was made by the company appears to be a major factor in transformation efficiency.