Another Problematic Ligation - 110bp ligation into 2.5kb vector (Jan/08/2006 )

I am attempting to ligate a 110bp fragment into a 2.5 kb vector. Both of which were double-digested with NcoI and HindIII (compatible buffer) and gel purified via commercial kit. I have verified the fragments subsequent to RE digestion by gel phoresis. The vector has been dephosphorylated (CIAP) after initial attempts of ligation without dephosphorylation. No colonies form after transformation. Ecoli are competent with control plasmid.

These are questions I have after combing through the forum archives:

How important is the molar ratio?

If 1:1 is the optimum ratio is straying from this going to result in absolutely no colony formation?

If I use a rapid ligation kit is it still advantagous to incubate the reaction overnight at 16C?

How crucial is the time for ligation versus length of the overhang?

Is it possible that exonuclease activity may have affected the insert or vector?

hi
optimal ratio for ligation is usually 1:3 to 1:5 (vector:insert). Using a quick ligation kit allows you to transform bacterias whithout let the reaction overnight at 16°

For the possibility of exonuclease activity, i'm not sure because as you gel purify bith fragments, contamination would occur from the elution buffer or the water you use to resuspend DNA. That's not the critical point...

Fred 33,
I appreciate your advice. I am currently using a rapid ligation kit and proceeding directly to the transformation. I was advised to leave the ligation at 16 C overnight and try ratios from 1:1 to 1:10. I am going to try again.

Thanks,
Wstore1

How are you purifying the vector after CIAP treatment? I believe that CIAP is hard to remove and requires either gel purification, phenol extraction or using a kit such a Qiaquick. Alternatively, something like anatarctic phosphatase (NEB) or Shrimp alkaline phosphatase (Roche) are excellent substitutes. Both enzymes are heat inactivated and no further purification is necessary.

Also, how is the 110 bp fragment generated? Is cut from a vector or PCR amplified?

ML1975,

I am using dephosphorylating immediately after RE digest and am gel purifying the reaction with PureLink silicon column extraction kit. It should be removing the proteins. As for the 110bp fragment it was grown up in a Topo vector and cut from it. THanks for your reply. If this helps you identify any problems, please let me know.
Thanks,
wstore1

My feeling is its the CIAP. I have never had success with it, so if you can get your hands on some shrimp alkaline phosphatase I would give that a go. The other thing I would try is to add heaps more 110 bp fragment to your ligation. Apart from that, your methodology appears sound to me. How competent are your cells?

quote name='wstore1' date='Jan 11 2006, 01:40 PM' post='36676']
ML1975,

I am using dephosphorylating immediately after RE digest and am gel purifying the reaction with PureLink silicon column extraction kit. It should be removing the proteins. As for the 110bp fragment it was grown up in a Topo vector and cut from it. THanks for your reply. If this helps you identify any problems, please let me know.
Thanks,
wstore1
[/quote]

I have performed thousands of ligations using a 100 bp insert.

I prefer using SAP during the restriction digest of the vector if the restriction enzymes are active at 30 - 37C. This avoids extra handling and purification steps which may lead to loss of material.

While I used CIAP and BAP for more than 10 years, they are frequently contaminated with exonucleases and you must use exactly the number of units/number of ends of DNA that each supplier defines. Simply adding a uL of CIAP to the reaction is probably going to result in damaged ends.

Alternatively, your DNA may have been damaged during UV exposure when cutting bands out of gels.

The optimum insert:vector ratio is 5:1 or 10:1 and is measured in pmoles or fmoles rather than ng. A ratio above 10:1 has a risk of generating double inserts into single-cut vectors.

While rapid ligation kits perform well, you will usually generate more colonies/ug by ligating longer.

tfitzwater,

I think you hit the nail on the head. Last night I tried several different molar ratios and left the reaction to incubate overnight. I have colonies now. I need to make sure that they have the correct insert. But, It seemed that using a greater ratio of insert to vector and leaving it longer helped. I will try the other phosphatases next time though.

Thanks everyone,
wstore1