Electroporation and arcing - (Jan/17/2008 )
Hello!
I'm having lots of trouble with arcing in my samples.
This is my protocol:
16s PCR product is ligated using TA cloning kit, recipe as follows:
-1 ul of a 300 mM, 15 mM MgCl2 salt solution (diluted 4 fold from the kit)
-1 ul of TOPO vector
-4 ul of PCR product
I add 2 ul of this ligation mixture to 50 ul of electrocompetent cells (thawed on ice, of course) and electroporate in pre-chilled cuvettes at 1.75 kV, 200 ohms, 25 uF).
I've been careful to avoid bubbles in the chamber, and have often watered down my ligations. I have a feeling that my cuvettes may not be cold enough - they seem to be sufficiently cold, but I have no idea of how long I should be leaving them on ice!
Any help is much appreciated! 
Consider drop dialysis of your ligation prior to electroporation. Float a millipore VSWP0025 membrane on DI water in a petri dish, shiny side up. Pipet your ligation mix onto the membrane, cover with the petri dish lid and let it sit for an hour. Pipet the solution off the membrane and use for electroporation. I would use only 1 ul. Practice this before doing it with a real sample, since pipetting onto and off of a floating membrane is a bit tricky. See http://www.millipore.com/techpublications/tech1/PF723
I don't even pre-cool my cuvettes at all and have no problems with arcing so I doubt that is the problem. I agree with phage434, give the dialysis a try. 
I'm having lots of trouble with arcing in my samples.
This is my protocol:
16s PCR product is ligated using TA cloning kit, recipe as follows:
-1 ul of a 300 mM, 15 mM MgCl2 salt solution (diluted 4 fold from the kit)
-1 ul of TOPO vector
-4 ul of PCR product
I add 2 ul of this ligation mixture to 50 ul of electrocompetent cells (thawed on ice, of course) and electroporate in pre-chilled cuvettes at 1.75 kV, 200 ohms, 25 uF).
I've been careful to avoid bubbles in the chamber, and have often watered down my ligations. I have a feeling that my cuvettes may not be cold enough - they seem to be sufficiently cold, but I have no idea of how long I should be leaving them on ice!
Any help is much appreciated!
Hey Jae,
Were you at UW-Madison years ago? We might know each other.
You can't use 2 microliters of a ligation in electroporation - there's too much salt. It will arc (as you have seen). Either use 0.1 microliters, or dialyse like the other post suggests.
Also, I assume you have added ligase ... your post doesn't mention it.....
I'm having lots of trouble with arcing in my samples.
This is my protocol:
16s PCR product is ligated using TA cloning kit, recipe as follows:
-1 ul of a 300 mM, 15 mM MgCl2 salt solution (diluted 4 fold from the kit)
-1 ul of TOPO vector
-4 ul of PCR product
I add 2 ul of this ligation mixture to 50 ul of electrocompetent cells (thawed on ice, of course) and electroporate in pre-chilled cuvettes at 1.75 kV, 200 ohms, 25 uF).
I've been careful to avoid bubbles in the chamber, and have often watered down my ligations. I have a feeling that my cuvettes may not be cold enough - they seem to be sufficiently cold, but I have no idea of how long I should be leaving them on ice!
Any help is much appreciated!
Hey Jae,
Were you at UW-Madison years ago? We might know each other.
You can't use 2 microliters of a ligation in electroporation - there's too much salt. It will arc (as you have seen). Either use 0.1 microliters, or dialyse like the other post suggests.
Also, I assume you have added ligase ... your post doesn't mention it.....
Nope I wasn't at UW-Madison!
I disagree that I am adding too much salt, as other people in the lab use the same protocol with no issues - the salt concentration is diluted 4 fold from the kit.
I am not using ligase, this is a TA TOPO cloning kit, the vector and insert join based on a long AAAA tail on the insert and many TTTTTs on the vector.
Anyway, thanks for you help!