Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Protocols and Methods Forum
Register Search Today's Posts Mark Forums Read

Protocols and Methods Forum Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.

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.

LinkBack Thread Tools Display Modes
Old 12-23-2003, 11:46 AM
Merijn Salverda
Posts: n/a
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 Salverda
Laboratory of Genetics
Wageningen University
Arboretumlaan 4
6703 BD Wageningen
the Netherlands
Reply With Quote
Old 12-23-2003, 05:51 PM
Posts: n/a
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.

"Merijn Salverda" <[Only registered users see links. ]> wrote in message
news:ebdb32ec.0312230346.3f5a83e2@posting.google.c om...

Reply With Quote

efficiency , high , transformation

Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
Human Cytome Project - Update 24 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 1 08-01-2010 02:18 PM
>>>> LOWER CHOLESTEROL <<<< Janis Stroud Forum Physik 0 09-22-2009 04:39 PM
too high PCR efficiency trouble Petrusha Real-Time PCR and Quantitative PCR Forum 6 06-12-2009 05:46 AM
High transformation efficiency binary vectors? Kirsten Knox Arabidopsis and Plant Biology 0 10-31-2007 02:34 PM
Human Cytome Project - Update 6 Jan. 2005 Peter Van Osta Cell Biology and Cell Culture 0 01-06-2005 11:18 AM

All times are GMT. The time now is 03:44 AM.

Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2015, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.15711 seconds with 16 queries