HELP!! transformation problems

Keith Rand Keith.Rand at molsci.csiro.au
Tue Aug 11 02:15:19 EST 1998


In article <35C85569.62571B4 at chclu.chemie.uni-konstanz.de>,
fof1 at chclu.chemie.uni-konstanz.de wrote:

> For routine cloning, where maximum competence is not an issue, we always use
> the TSS method of Chung et al. 
> It is very reliable once you got it to work. Thats how I do it:
> 
> * grow bacteria in 10ml LB to OD=0.8 (we have better results at 0.8 than
at 0.4)
> * spin down for 5min at 8000g
> * decant supernatant
> * add 500ul of ICE-COLD LB, resuspend by vortexing
> * add 450ul of ICE-COLD 2xTSS (see below), and, simultaneously, 50ul of (RT
> warm) DMSO
> * mix by very gentle shaking (NO VORTEX!!)
> * add 150ul of suspension to the DNA in a prechilled microcentrifuge tube
> * mix by gentle mixing (AGAIN NO VORTEX!!)
> * let stand on ice for a minimum of 10min (usually 30min)
> * plate onto LB-agar plate with appropriate antibiotic, e.g. amp
> * grow overnight at 37C
> 
> Ok, there are some points to consider:
> * to make 2xTSS, dissolve 4.1g MgCl2 and 40g PEG-8000 (other PEGs also work)
> in 200ml LB. Sterilize by autoclaving. After autoclaving, there will be a
> phase separation as PEG settles. Mix the phases by shaking vigorously. The
> solution will be slightly turbid, but is ok. Turbidity will disappear after a
> few days. We store the solution at 4C where it is stable forever.
> * DO NOT make up the TSS solution 1x to directly resuspend bacteria. It simply
> does not work for whatever reason.
> THIS IS MOST PROBABLY THE REASON WHY IT DOES NOT WORK IN YOUR HANDS!!!!
> * it is better to autoclave than to put thru a 0.2um filter, because some
> filters contain detergents that will inhibit bacterial growth.
> * always use ICE-COLD LB for resuspending bacteria, and ICE-COLD TSS. DMSO
> must NOT be put on ice as it solidifies.
> * never use a vortex to mix the bacteria with LB or TSS
> * there is no need for a recovery period before plating, nor for glucose
> 
> Using this method, we routinely get a efficiency of 10e6 to 10e7. Never 10e8.
> This is sufficient for routine cloning, but NOT sufficient e.g. for library
> construction. Best method for high efficiency is electroporation, or the Inoue
> protocol for chemical competence.
> 
> Hope this helps
> Frank
> 
> PS: Do not try to freeze the bacteria. They will go down in competence at
> least a factor of 100. Well, that might still be ok for transformation of
> supercoiled plasmids, but does not work for ligations any more.

But Chung et al claim that they can freeze the competent cells with little
or no loss of efficiency. With the Inoue protocol, the efficiency
increases after freezing. Many labs use frozen competent cells, and highly
competent frozen cells are available commercially. Were you just unlucky
on one occasion, or can you never successfully store your cells? If you
could identify the reason for the loss of competency it might be very
useful for others. Has anyone else found that they can't make batches of
good, frozen competent cells?

-- 
Keith Rand,  Sydney Australia



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