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high efficiency transformation

high efficiency transformation - Protocols and Methods Forum

high efficiency transformation - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 12-23-2003, 11:46 AM
Merijn Salverda
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Default high efficiency transformation



Hi all,

I'm currently attempting to get high efficiency transformation of a 4
kb plasmid into DH5alfa (E. coli) using electroporation. I'm aiming at
an efficiency of 10e8 to 10e9, but so far I got stuck at 10e7. My
plasmid has a tetracycline-marker, and I recently came across an
article that reports lower transformation frequencies when selection
is made for tetracycline ('The result is a transformation efficiency
comparable to that of chemical transformation' - Hengen 1995). Does
any of you have any experience with this? And does anyone have any
suggestions how to get higher yields anyhow? I've been thinking about
ordering ultra-competent cells (e.g. Electromax DH5alfaE), but as I
need to do shiploads of transformations, this would make it a rather
expensive matter...

Thanks in advance!

Merijn.


Merijn Salverda
Laboratory of Genetics
Wageningen University
Arboretumlaan 4
6703 BD Wageningen
the Netherlands
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Old 12-23-2003, 05:51 PM
EK
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Default high efficiency transformation

Depends on how you prepare your super electrocompetent cells. We make ours
by washing freshly grown cells (OD600 not higher than 0.3) 5 times in cold
water (keep cells cold at all times), 1 time in 10% glycerol, and
resuspending in the minimal amount of cold 10% glycerol. Try to resuspend
cells in less than 1/100 volume of initial culture (volume of washes is not
important: 1/10 of initial or more is OK). If you do everything careful
enough, you will get at least 10^10 transformants per mg DNA. Don't forget
to pre-grow electroshocked cells in SOC for 40-50 min before plating.
Emir

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