5 replies to this topic

[swinhoe]

I am trying to ligate a 5118bp fragment to a 9850bp vector without success. Both fragments are gel extracted using Qiagen gel extraction kit, followed by phenol:chloroform. I’m using T4 DNA Ligase from NEB. I have tried varying insert:vector ratios and many competent cell lines including JM109, DH10B, StBl2 and HB101. My transformation controls always work so I don’t think the problem is with the transformation but possibly with the ligation reaction or toxicity. Can anyone offer any advice or help, it would be greatly appreciated. Thank you.

[Solomona]

Did you observe your ligation product on a gel?

[swinhoe]

No I haven't. I presume I should look for a band the size of the ligated product?

[yzhou715]

Please try this:

1, Make sure you washed with 500 PB buffer and let PE buffer set more than one min.
2, I would not use Phenol;chlorform after Qiagen kit purification.
3, de-P your vector with SAP.

Good luck!

[Solomona]

Yes, run a gel with the recombinant vector in one lane and also one with a cut at a single restriction site.

Try eluting the DNA from the column with preheated water (~45°C 2 min. or more) and transfer the eluate directly into the ligation reaction.

[tuckern]

You should SAP treat your vector (but make sure you kill it after). You probably don't need the phenol chloroform step if you've already gel purified your fragments. You'll just lose DNA.