exaggerated transformation efficiencies c
Ian A. York
iayork at panix.com
Mon Sep 8 08:09:28 EST 1997
In article <Pine.A188.8.131.520908051135.38022A-100000 at aix1.uottawa.ca>,
colossus... <s535290 at aix1.uottawa.ca> wrote:
>I have now encountered two occasions where commercially available
>"super-competent" cells have turned out to be no better than home made
>competent cells I have made using the one step TSS protocol. What in the
You don't say how much you do get out of them, but I'll give you the
dubious benefit of my uncontrolled and probably-irreproducible experience
anyway. I've tried a few different ultra-comp cells and have generally
found them to be pretty good--more than half the manufacturer's claim
(e.g. 3 x 10^9/ug vs the claimed 5 x 10^9), which I consider acceptable.
However, the results are heavily dependent on following *exactly* the
protocol the manufacturer gives. For example, when they say to dilute
your ligation 10-fold and use 1 ul per transformation while whistling "Ave
Maria" and sacrificing weasels with obsidian blades, don't try to
substitute a flint blade instead.
Also, the efficiency that they claim is usually only seen for specific
amounts of DNA. Once you get too low you can get a sudden drop-off, and
once you get too high you still get lots of colonies but the per-ug
efficiency goes way down.
The efficiency is also somewhat dependent on the quality of the DNA, but I
can't say I've found this a huge problem in chemically competent bugs.
With electroporation it does make a big difference; it's critical to get
rid of salt in the prep.
If you're doing the transformation with control DNA (most manufacturers
provide a control plasmid, which you should use) and following directions
exactly, and you still aren't getting close to the manufacturer's claim,
then you should be able to kick up enough fuss to get something back from
them, I would think.
Not much help, I suspect, but there it is anyway. Good luck.
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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