From: frist@ccu.umanitoba.ca ()
Subject: Re: Frozen competent RecA- strains
Organization: University of Manitoba, Winnipeg, Manitoba, Canada
Date: Thu, 10 Sep 1992 17:37:03 GMT

In article   writes:
>edu.au!monu6!vaxc.cc.monash.edu.au!mic236u
>From: mic236u@vaxc.cc.monash.edu.au
>Newsgroups: bionet.molbio.methds-reagnts
>Subject: Frozen competent RecA- strains
>Message-ID: <1992Sep7.124536.90018@vaxc.cc.monash.edu.au>
>Date: 7 Sep 92 02:45:36 GMT
>Organization: Computer Centre, Monash University, Australia
>Lines: 37
>
>Hi Fellownetters,
>
>Some time ago I read somewhere that competent RecA- cells don't like to be
>frozen; they lose their competence in a couple of weeks.
This is a myth that is almost  as old as the idea of freezing competent cells.

 At the time it 
>explained the problems I had with my frozen competent cells. (Unfortunately, 
>I did not keep the reference.) 
>
>When I came in this lab I found that here every one or two months a fresh 
>batch of competent cells (DH5alpha) is made and stored at -70C.
> This works 
>fine most of the time. However, recently we had problems getting 
>transformants from ligations whereas cccDNA worked fine. 
>I hope a fresh batch of competent cells will solve our problem.
>
>But some questions are inevitable:
>
>- Has anyone out there the reference I forgot to keep?

I don't know what paper you're referring to, but the most comprehensive
study on transformation that I have ever seen is

Hanahan, D. J. (1983)  Mol. Biol. 166:557-580.

This paper exhaustively studied many parameters influencing transformation,
including treatment of cells, size of DNA, strain variation, and many more.
 
>
>- Can cells lose competence for oc/ccDNA but stay competent for cccDNA?
>  I know: transformation efficiency cccDNA >>> oc/ccDNA. But we got good 
>  numbers of colonies with cccDNA and zero with several ligations, and
>  the ligations worked! What is the difference in the uptake of cccDNA 
>  and oc/ccDNA; is it just a matter of size? 
Hanahan showed that "the relaxed form of each plasmid [tested] transforms
about 75% as well as its supercoiled form" ie. not significantly different,
for most practical applications. It is possible that with other
strains, or other protocols, this relationship would not hold true.
However,  before you can conclude that in your case cccDNA >>> oc/ccDNA,
you need to do the proper controls. These might include: 1) put your cccDNA
through exaclty the same ligation reaction as your test DNA. 2) Try nicking
the cccDNA  3) add some of your ligation mix to the cccDNA.

One thing that can often result in decreased efficiency is adding too large
a volume of DNA to your competent cells. Some protocols recommend that the
volume of the DNA should not exceed 1/20 the volume of the competent cells.
Otherwise, you may change the concentrations of important components in
the cells, or add too much of some component (eg. a cation) that could
inhibit transformation.

>- Has anyone a good method to keep RecA- strains competent for a long 
>  time at -70C? Some companies sell frozen competent RecA- cells. For 
>  how long are they guaranteed to work? 
In my hands, comptent cells retain their transformation efficiency for 
at least a year, if stored properly (see below).

The Hanahan paper introduced DH1, the predecessor of DH5alpha. Both are
recA- strains. Hanahan saw no difference between recA+ and recA- cells,
w.r.t. transformation efficiency.

There are two things that frequently ruin frozen competent cells. First is
storage. If  cells are allowed to warm up (folklore says >-40C but I have
no hard data on this) and then re-freeze, their transformation efficiency
goes down. The temperature guage on your -70 freezer can be misleading. All
it really indicates is the temperature in the part of the freezer
immediately adjacent to the probe. If the freezer is open for a long
time, or if a massive, warm object is placed next to your cells, that
object cools down by distributing its heat to the things in its immediate
vicinity ie. your cells! The take home lesson here is that you should take
extra measures to isolate really critical things in your freezer (eg. comp.
cells, antibodies) from other items that can tolerate some warming.

The second factor that often ruins competence is thawing prior to use.
Hanahan recommends rapid thawing at room temperature, as opposed to
gradual thawing on ice.  What I usually do is to take the tubes out
and lay them on the bench top, checking every few minutes to see whether
the outer layer of ice has melted. When it has, you can usually tap it
a few times and the rest of the ice will turn liquid. Immediately place
the now liquid cells on ice.  

If you are useing a transformation protocol that requires heat shock,
remember that the time of the heat shock needs to be optimized for
the volume of the sample and surface area of the tube. This is because
the rate at which the sample warms up, as well as the time it stays at
42C, will differ with volume and surface area, and perhaps the composition
of the tube (heat transfer may vary between polypropylene, polystyrene,
glass etc). If the manufacturer of your cells recomends a particular volume
and type of tube, stick with that.

I'm not sure how important the method of freezing is. If you make your own
cells, it is probably safest to freeze in liquid N2 or in a dry ice/ethanol
bath, rather than to let them freeze slowly in the freezer. 
>Cheers Volker

===============================================================================
Brian Fristensky                | Unintentionally funny qoute of the month:
Department of Plant Science     | 
University of Manitoba          | Rev. Jerry Falwell denounces Bill Clinton,
Winnipeg, MB R3T 2N2  CANADA    | saying that his "misquoting and manipulating
frist@ccu.umanitoba.ca          | holy scripture for political purposes should 
Office phone:   204-474-6085    | be offensive to millions of Americans." 
FAX:            204-261-5732    |
===============================================================================