washing electroporation cuvettes - can't get rid of contaminants (Nov/24/2005 )
i gel purify my plasmid and insert. They both look fine.
I do a ligation with new enzyme, new buffer, new tips, took colleagu bacterias and plates (thanks to him)... I have clones
i've already checked 90clones and no one is positive.I've tried to analyse restriction digests and i'm surprised all is other vectors i've cloned recently.
I'm tired to waste time and material screening contaminants.
Since new cuvettes won't come till next year ( ) may you share your procedure for cleaing electroporation cuvettes?
thanks a million
Hi, I can't find the protocol I used when I had to wash my cuvettes (thankfully I don't have to do that anymore) but I remember that I would first rinse them with water, then if I remember correctly let them stand in HCl (1M? ) for 10 or 15 minutes, rinse again with ddH2O and finish with ethanol to dry. I never had contaminants with this protocol (while I had some with a gentler one). BUT your cuvettes won't stand much cleansing and need to be changed after a few times with this treatment.
You might try a wash with 3% hydrogen peroxide. HCl is a good choice for eliminating the DNA, but will likely corrode the electrodes quickly. I doubt if it needs to be 1M. 10 mM probably works as well. I think you can determine a good method experimentally pretty easily by adding plasmid DNA, cleaning, and electroporating cells without any additional DNA.
I'd like to hear what you eventually settle on.
thanks for your replies. I'll try to wash my cuvettes with NaOH. I think it's less corosive than HCl too.
Well dear phage434... I must confess my english is quite poor. May you explain me part of your last sentence : "what you eventually settle on".
I've undertood you want to know if i succeed to wash... I'll try to add 1µg DNA. Swirl. and let sit 30'. Then wash my standard procedure (water then EtOH) in comparison to 3% NaOH and see if any colonies grow.
Am i correct?
Yes, I want to know what works. Your English is better than any attempt I might make at any other people's native language.
I think acid is more effective than base at destroying DNA.
I usually wash electroporation cuvettes with water, and rinse again with ddH20, dry them in sterilizing environment,eventually store them at 75%ethonal,if I wanna make electroporation,i will dry them again before use.This treatment works well in my lab.