Problem with Ligation Controls HELP PLEASE - EcoR1 cut site on pEGFPC2 vector will not religate? (Sep/23/2008 )

Hi,

I am trying to set my controls for ligation of 1.9kb insert (EcoR1/BamH1) into pEGFP vector.

1) To make sure ligation is working, I have used EcoR1 to digest my pEGFPC2 vector --> I run a gel to RE digest products parallel with uncut vector, and I can see clearly that the vector is cut....

2) I cut out the digested vector and do a gel purification.

3) I then use T4 ligase O/N @ 16C. After transformation, the ligation product does not form colonies on Kanomycin resistant plates, however my uncut pEGFPC2 vector does form colonies.

I have repeated this simple ligation with both NEB and Invitrogen ligase enzymes - and I have no luck.

*** When I run the ligation product on a gel, I am not seeing much show up, so I am thinking that I am loosing a lot of my vector in gel purification??? I use the Quiagen kit.

Anyone have any ideas of whats going on here?? Im new to this.

in my case, i usually add other control, which is a cut vector with no insert. Normally you would still see some colonies due to the fact that your vector is not really 100 pct cut. At least this may give you a clue if you are losing your vector in the gel purification.

or maybe, its an insert problem. how about your insert to vector ratio?

but that is because i dont gel purify my cut vector. It works for me that way

1) I assume you also digest it with BamHI, right?

3) Obviously your vector were being digested thoroughly. So you should focus on your insertion. What is the source of insertion? Is it a PCR product or from other vector?
If it is from other vector, then check for vector:insert ratio (in ng, not in volume)
If it is a PCR product, you can try to use TOPO cloning kit. Ligate the PCR product into TOPO. After transformation, digest the recombinant TOPO vector with BamHI and EcoRI. Then I can ensure you have a DNA fragment with BamHI and EcoRI RE site ends after doing gel purification. Use the gel purification product for your ligation.

***From your words, i felt that you did not make DNA concentration measurement. It is really needed as a beginner. Besides that, a ligated product mostly cannot be visualized using gel because its concentration is too low, however it is sufficient for transformation.

Please let us know when it works. Thank you.

I will be using a 3:1 vector : PCR Product ratio ...

BUT!!! the thing is.... I am not using my insert yet, because the controls are still not working.

I have been introducing 1 cut (EcoR1) into my pEGFPC2 vector and then trying to ligate it back in order to allow for colony formation.

I have been using the Invitrogen T4 Ligase / buffer kit - and I can't seem to get a ligation of even the single cut vector (No insert)

My uncut vector allows for colony formation - the ligation product does not

Sounds like it's a problem with your DNA gel extraction - look at the back of your qiagen handbook for possible troubleshooting - possibly left over ethanol on the membrane or salt concentration is too high - this can affect ligation efficiency. keep a before and after sample of your digest so that you can gauge how much DNA you're recovering during the gel extraction. Use a DNA ladder to judge how much you have in your sample after recovery - spec readings are often inaccurate after gel purification. Also heating the elution buffer to 55C often helps you recover more product.

A quick way to test if your ligase is working. Take some DNA ladder and try to ligate it to itself. You only need to do the reaction for 30 minutes at RT. Then, run a gel. If your DNA ladder has shifted to higher sizes, the ligase is OK. If nothing happens, get some more ligase.