transformation problems

Dr. Paul N. Brothers brothers at umbc.edu
Wed Mar 20 18:23:35 EST 1996


GILLIAN NI BHARLO BOTANY PG (NIBHARLO at acadamh.ucd.ie) wrote:
: Hi there!
: I'm hoping someone can explain what I've done wrong during a recent 
: transformation. I was transforming a ligation reaction and also 
: retransforming an old plasmid. The volume of the ligation was 40ul, 
: which I added to 200ul of cells (normally I add 20ul ligation mix to 
: 200ul cells). For the plasmid retransformation I just added a couple 
: of microlitres. After incubation on ice and heat-shock at 42 added to 
: medium, 37 shaking for about an hour (normally aboout 40 mins but I 

I do not shake, rather let them sit at 37 for an hour and a half
before plating.  Competant cells which have just been transformed
are very fragile.

: forgot about them). The cells in the ligation mix transformation had 
: aggreggated as though lysed, those in the retransformation were fine. 
: Was the increase in ligation volume the problem or were they just 
: left too long? I had increased the ligation volume in a desperate 
: attempt to get a problematic TA subcloning to work....

Did you run an agarose gel of your ligation mix to see if ligation
is working?

Did you try ammonium acetate precipitation of your ligation mix?

Alternatively, did you try Gene Cleaning your ligation mix?

Did you prepare your own compentant cells or use commercially
available ones?  If you prepared your own,
sometimes the detergent you use to clean your glass and
plasticware used to prepare the competant cells can reduce efficiency.
Autoclave all reuseable glass and plasticware several times in 
fresh changes of double distilled or distilled deioninzed water.  Paul.



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