Ligation with EcorV and PacI - Ligation and transformation! (Nov/25/2010 )

Hi

So I am trying to ligate a 3.3kB insert into a 15.9kB plasmid. The plasmid is a pBR322 with a viral genome inserted into it. Both the plasmid and insert have been cut with EcorV(blunt end) and PacI (cohesive end). I purified using the QiaexII kit and did a ligation reaction using T4 DNA ligase in a 20uL reaction. I used different ratios of insert to plasmid. Left the reaction overnight and I transformed TOP10 cells the next day with 4uL of the ligation.

I don't have any colonies the next day...I checked the plates I used and its ampicillin and the bands on the gel after gel purification were really clean. I had a good amount too, so I have no idea why it didn't work.

If anyone has any experience with this please let me know it would be much appreciated.

What is the transformation efficiency of your cells? They will need to be high efficiency for this to work. I would recommend testing this by transforming your parent 15 Kb plasmid, since it will test transformation of large plasmids with the same resistance and origin as the ones you are trying to build. You should aim for > 1e8 cfu/ug of plasmid DNA, either with chemically competent cells or with electroporation.

You should also try a crowding agent such as PEG in your ligation reaction, or simply use the quick ligase buffer and follow the directions for blunt end ligation.

Hi
Yeah I always use my parent 15kB plasmid as a positive control for transformation and I get A LOT of colonies. I have never used PEG but the ligation reaction is definitely an issue since I ran my ligation reaction after I let it incubate overnight at 14degress and I didn't see a single band.
I am not sure what else I can change in order to get the ligation to work.

I would do this by the number, and check each and every step

1 - after restriction digest, gel purify vector and insert
2 - verify that you have the appropriate digested insert by gel
3 - verify that you have the appropriate digested vector by gel
4 - quantify vector and insert using a nanodrop.
5 - ligate vector. and insert. use a mol ratio of 1:1
6 - run a vector control (aka the double digest vector that you have without adding the insert)
7 - run part of the ligation mix onto a gel. You should see high molecular weight bands. Also run part of (6) on a gel. No high molecular weight bands should be seen here.
8 - transform ligation mix into supercompetant cells. Do the same for the vector control

And yes, do try the PEG.

Thanks, I feel a little better that I am not doing something completely off the mark.
So i have been doing that by the number. My gels are really clean and I have the the size that I want for both the vector and the insert and I have quantified using the nanodrop and I end up getting really good amounts. But when I run the ligation mix onto a gel I don't get high bands. Its the wierdest most frusterating thing. I have used competent cells that I have made in Lab and I also used TOP10 competent cells from invitrogen and NEB5-alpha cells. My vector of 19.2kB that I use for a positive control has a lot of colonies in all three of those cell types.
Is the PEG really that important, No one around me seems to have it and I don't think I need to crowd since the vector transforms fine without it etc.

You can debug ligations by ligating just the vector DNA and (independently) just the insert DNA.
You can then look at the ligated and unligated results on a gel. For doing this, I would use ordinary ligase buffer and heat kill the ligation reaction before loading on a gel.

You should see double length fragments in both cases (and ideally, longer fragments).
If you do this with one or the other enzyme in the ligation mix, you can figure out which is ligating and which is not.

The role of PEG (or equivalently, quick ligase buffer) is to allow the blunt ligation to proceed more rapidly and efficiently.

You might want to check the ligase buffer. It can go bad (has ATP in it).

Ligase buffer contains the reducing agent DTT. Thus it has a very strong smell. If the buffer doesn't smell, the DTT has been oxidized and the buffer has gone bad.

It is a useful thing to aliquot the ligase buffer.

My ligation is ok because I have positive controls for my ligation. I cut my plasmid individually with the two restriction enzymes and ligate and transform to see if my vector religates which it does.

I was working with TBEV virus, sometimes the colonies appear after longer incubation. So trying leaving the plates for longer in the incubator and see if you get colonies..