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Double restriction digest, ligation, transformation

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Double restriction digest, ligation, transformation

Postby Bsalterton » Thu Feb 12, 2009 12:52 pm

Hi,

I have a couple of problems with a ligation + transformation...
I have amplified a 6kb fragment and want to clone it into a plasmid which is 5.5kb. The plasmid that I am using has already got an insert in which I am removing by a double restriction digest using XhoI and ApaI.

(1) Ideally I wanted to then run the vector digest on a gel and gel extract so that I remove the insert already there, but I cannot get anything more than 5% yield after using the Qiagen gel extraction kit. I have looked on numerous forums and have recently found a link that suggests that the components to the QG buffer in the kit changed and didnt work?? has anyone else heard of this? We have tried this so many times tweaking the protocol and no luck.

(2) So because I was unable to gel extract, we decided to go for it and use neat PCR product and Vector, perform the double digest and then screen many many colonies in the hope of getting just one which contains my insert and not a religated plasmid! However, after performing the ligation and transformation yesterday....I have found this morning NO COLONIES!

I was using a fast ligation kit (Roche) which had only a 5minute incubation time and a novablue giga single competent cells.
- Could the incubation time been too short?
- Are these cells capable of holding a plamid of 6+5.5 kb?
- Or is it that the double digest was incomplete and I ended up with just linear DNA and so cells could not grow on the AGAR + AMP plates?

NB: I only did a negative control as these cells have been used previously and known to work...although who knows what could have happened in the past few weeks!!

Any suggestions as to how I should proceed would be appreciated!!
Bsalterton
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Postby nena77 » Thu Feb 26, 2009 10:10 am

If I remeber correclty the Quiagen gel extraction kit has different color that tells you if the binding of the DNA to the memebrane works correclty....I had a similar problem and I remember that an imporant step in ensuring the correct binding is the addition of isopropanol, because it adjusts the pH. I think it is also suggested in the kit. I had also very little amunt of DNA afer elution because my plasmid was bigger than 10.0000 KD ( the kit is fdesign for smaller plasmid). However, you can cut the band as small as possible, and use a little volume for elution. I always digest my plasmid in parallel tubes and I pooled the eluted DNA. Another great approach that helped me was to elute the amount of DNA in 25 ul of warm eluton buffer of several tubes and then centrifuge the pooled purified plasmid using the protein concentrator, microconcentrifugal filter devices YM-100 from Millipore. It is normally used for protein but it word also for DNA. You centrifuge and then resuspend your plasmid in little volume. If you are not sure about the solution in the kit order a new one. The QUIAGEN provide a fre small kit for gel extraction for 10 samples.

I normally perform the ligation overnight, at 4 degree. Which kind of cells did you transform? Because maybe the transformation is did not work at all, rather than the ligation.

Elena
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