Electroporation Arcing - I'm a newbie, any pointers welcome (Feb/18/2010 )
Hello all,
I am in the process of creating several constructs to study the interactions between a recombinant ubiquitin-like protein and recomb. viral protein. I have used other means of transfecting E. coli before, but I'm new to electroporation. I'm also in charge of writing a protocol for the lab handbook. I would appreciate any technical notes offered.
I have a Harvard Apparatus ECM 399, and I am transfecting 40 ul of XL1-blue E. coli with 2 ul of vector (after SAP dephosphorylation) and 1 ul of RE digested PCR product at 1370 V (my advisor's suggestions). Each time I initiate electroporation, I see a visible arc and hear a quiet 'snap'.
The arc doesn't seem to affect the viability of the bugs, since I have gotten colonies from my last 3 transfections. The owner's manual suggests changing the transfection volume, purifying the DNA, etc.. I am going to purify my PCR product before I do my next transfection. I have a few questions, though:
Should I be concerned about the arc if I still get colonies?
Would changing the freezing medium of the host help prevent arcing? I'm not sure what the E. coli have been frozen in--I'm new to this lab--but I'm guessing Miller's LB and glycerol. Would switching to Lennox lower the salt concentration enough to avoid the arc?
Thank you!
lab rat
Are you making the cells electrocompetent before electroporating? Are you ligating the insert to the vector? Are you drop dialyzing after ligation?
Are you making the cells electrocompetent before electroporating?
Yes, but someone else did it for me. The lab keeps them in the -80 until needed.
Are you ligating the insert to the vector?
Yes. I wasn't very clear on that, was I? I double-RE digest my plasmid, then dephosphorylate with SAP. I also digest my PCR fragment, and ligate with T4.
Are you drop dialyzing after ligation?
No. My advisor said it's not always necessary, and to try adding the ligation mix to the cells and zap them. If that didn't work, then drop-dialize using a membrane and water. That's what I plan on doing next.
lab rat on Feb 19 2010, 09:58 AM said:
Yes, but someone else did it for me. The lab keeps them in the -80 until needed.
Are you ligating the insert to the vector?
Yes. I wasn't very clear on that, was I? I double-RE digest my plasmid, then dephosphorylate with SAP. I also digest my PCR fragment, and ligate with T4.
Are you drop dialyzing after ligation?
No. My advisor said it's not always necessary, and to try adding the ligation mix to the cells and zap them. If that didn't work, then drop-dialize using a membrane and water. That's what I plan on doing next.
Drop dialyzing should help. It may not be necessary if using a small enough volume of the ligation in the electroporation, but it's a relatively easy step. I've read it can be done in 10 min, but the one time I tried it at 10 min, it arced, so I always go at least a half an hour.
My guess is the dialyzation will clear up the issue.