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after transformation - (Aug/02/2007 )

Dear aLL,
I did ligation and ligation control (only with cut open vector). Then I transformed the ligation mix and ligation control into E.coli. Then I did blue/ white screnning. But after one night, 37oC, I found that only 4 white colonies, no blue colonies on ligation mix plates. 5 white colonies, no blue colonies on ligation control plates. And I also transformed uncut plasmid into E.coli. It gave very good results. The siye of colonies were also big.
so does anyone know why the ligation mix plates gives so little colonies?

btw, there is a very very very thin layer of cells on the agar plates. White colonies grow on the layer.


-Lucy Wong-

Hmmm... are the ends of your vector compatible, @can the vector self ligate? Did you do a dephosphorylation on the vector? Dephosphorylation prevents/decreases vector religation.

Nevertheless, I can think of a few possibilities for the observation,
1- I am not sure how you transform your cells. But if you when via electroporation, you would have had to percipiated your ligation mix. It is possible, that you lost your ligated DNA at point. I would recommend that you run a portion of the ligation onto gel after resuspension in water (just prior to transformation) to make sure the DNA is still in there.

2-the ligase and/or the ligase buffer no longer work. Both ligase and ligase buffer go off fairly easily. So have any one done a sucecessful ligation with the lab's ligase recently? Try getting hold of some ligase that is know to work from the labs nearby. Same thing with the buffer. Neither Ligase or the buffer stand freeze thaw cycles well.

3- If dephosphorylation was conducted. Overdephosphorylation may have damaged the vector's overhangs. Could you tell us how much DNA was dephos, for how long, with how much CIP, in what buffer and at what temperature. How did you deactiva the phosphotase. Rule of the thumb: 1pmol DNA will dephosphorylated by 0.1Units CIP is 1hr at 37Celsius within a volume of 50ul in NEB buffer3.

4- the ligation you are attempting is rather difficuly. (eg 12kb vector + 8kb insert) Your cells aren't good enough to get over the hill. I would recommend genehogs (oneshot electrocompetent cells) from invitrogen.

I assume you are recovering your cells in SOC for at least 30mins at 37 celsius before plating them onto the selection plate. The cells need time to recover, and start expressing the resistent gene.