Electroporation cuvettes
Cristina Cebrian
Dear neters,
Does anyone know the best way to clean the electroporation cuvettes?
I've tried autoclave and UV light but seems it's not enough.
Thanks in advance,
Cristina Cebrian
ceb...@ar.vhebron.es
Centre d'Investigacions en BQ i BM, Hospitals Vall d'Hebron.
Plta.14 Hospital Materno-Infantil.
P§ Vall d'Hebron 119-129.
Barcelona.
Kevin Mulcahy
Dear Cristina,
I asked the same question last week and got some useful replies both via
the newsgroup and via email. Here are the replies:
Matthias Vogel wrote:
"I always reuse my electroporation cuvettes for now more than two
years without contamination problems.
After transfection I soak the cuvettes in Water with washing-up
liquid overnight. Then I clean them with pipe cleaners and rinse
them thoroughly with a hard jet of water (deionized). Then I put them
in 70% EtOH overnight.
Before using dry the cuvetts upside down in a sterile bench."
Eric Anderson wrote:
"this is another one of those "search the archives" type questions. there
are a bunch of different ways to clean electroporation cuvettes (probably
almost as many cleaning methods as there are investigators who can't
afford to just throw them out). even within our lab of 10 people there
are ~ 4 different methods. here are some basic do's and don'ts.
Do:
- get them in mQH2O asap after you finish using them.
- rinse them 4-20 times (different strokes for different levels of
paranoia) with mQH2O.
- rinse with either 70% or 95% EtOH (we usually use 70% then a couple more
water washes then another 70% followed by a 95% to dry them)
Do Not:
- use bleach.
- autoclave.
- wait more than overnight to clean them completely after your
experiment. i prefer that they get cleaned immediately after the
experiment but a couple of lazy students in this lab seem to get away with
letting them go overnight in 70% EtOH.
as for sterilizing, we just stick them in a Stratalinker standing up and
set the power to 5000. in my old lab we used to put them on the UV
lightbox for 15 minutes."
Dominic Voon wrote:
" We too do a lot of electroporation. We use the BIORAD GenePulser
set-up, it wasn't long before we find that at $3 a pop these cuvettes
will cause major budget blow-out. We actually read off the discussions
in this newsgroup that one could wash a used cuvette thoroughly in
distilled water (to get rid of the salt) and submerge it in 70% EtOH till
the next time (I dry them in a sterile hood till I am ready to put the cell
suspension in them). We have since been doing it with no apparent problem.
Give it a try - there are more paranoia ways of going about it, but we find
that this method is good enough."
John Watson wrote:
"In my old lab we would always re-use electroporation cuvettes. Washing
was done in water, distilled water, and finally 70% ethanol. I would
sterilize prior to re-use by UV irradiation. Note that cuvettes cannot be
reused indefinitely -- eventually they will arc and that will be the end of
that."
Hope these messages help.
Best wishes,
Kev.
Kevin Mulcahy
Chris Boyd
Cristina Cebrian (ceb...@ar.vhebron.es) wrote:
: Dear neters,
: Does anyone know the best way to clean the electroporation cuvettes?
: I've tried autoclave and UV light but seems it's not enough.
Blimey heck! If you see contamination after autoclaving AND UV light
treatment, then I suspect your source of contamination is elsewhere.
I'm surprised cuvettes even survive autoclaving. It's not unheard of
for batches of electrocompetent cells to be contaminated with plasmid
DNA. Of course this only shows up after electroporation, so plating
out some unzapped cells is not a good enough control. Most likely
sources of contamination of this kind are aerosols from contaminated
Gilson barrels and the like. Either use plugged tips, or regularly
clean out (and/or autoclave) Pipetman barrels.
It's just occurred that you might be complaining about arcing rather
than contamination after autoclaving/UV light. There are lots of ways
to clean out cuvettes to avoid this, see the FAQ list. Personally I
flush out the cuvettes vigorously with copious amounts of hot tap water
immediately after zapping, then store in 100% EtOH for at least 24 h
before drying out in a laminar hood. Then UV treatment if I remember.
Throw out any that exhibit crazing.
Best wishes,
--
Chris Boyd | from, | MRC Human Genetics Unit
chr...@hgu.mrc.ac.uk | not | Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb | for | Edinburgh EH4 2XU, SCOTLAND
Richard P Grant
Chris Boyd (chr...@hgu.mrc.ac.uk) gibbered:
} Cristina Cebrian (ceb...@ar.vhebron.es) wrote:
} : Dear neters,
} : Does anyone know the best way to clean the electroporation cuvettes?
} : I've tried autoclave and UV light but seems it's not enough.
} Blimey heck! If you see contamination after autoclaving AND UV light
} treatment, then I suspect your source of contamination is elsewhere.
Possibly the autoclave. Don't laugh - plasmid DNA survives autoclaving
remarkably well. The autoclave must be the most efficient method of
spreading ccDNA around the place known to man. When we make competent
cells, the glassware we use does not go anywhere near the autoclave or the
washing up machine.
--
Richard P. Grant University of Oxford
Nuffield Department Obstetrics and Gynaecology FFPGP
http://users.ox.ac.uk/~lady0266 http://www.molbiol.ox.ac.uk/~rgrant
---------------------# hit any user to continue #---------------------
brett
>rgr...@worf.molbiol.ox.ac.uk (Richard P Grant) wrote:
>
>>Possibly the autoclave. Don't laugh - plasmid DNA survives autoclaving
>>remarkably well. The autoclave must be the most efficient method of
>>spreading ccDNA around the place known to man. When we make competent
>>cells, the glassware we use does not go anywhere near the autoclave or the
>>washing up machine.
>
>bleach or UV or a combination? Or something I haven't thought of?
Bake
Brett Lindenbach
Program in Immunology
Washington University - St Louis
br...@borcim.wustl.edu