cloning problems

Ian A. York iayork at panix.com
Tue Nov 10 11:30:26 EST 1998


In article <36485255.43925A4D at gengenp.rug.ac.be>,
Esbjorn Fiers  <esfie at gengenp.rug.ac.be> wrote:
>
>1. Is it necessary to inactivate the
>restriction enzymes after the digest,
>before electrophoresis?

No.  

>2. What is the equation for determining
>the composition of a ligation? I mean an
>equation that gives me the quantity of
>each fragment (in relation to its
>length) that should be added.

If you have cohesive ends, I aim to have a 3:1 insert:vector ratio.  If it
is a blunt-ended ligation, I aim higher--10:1 or so.  I also usually
include a dilution on either side of my target ratio, so for example I'll
set up ligations at estimated 1:1, 3:1, 9:1 for cohesive ends.

This is a molar ratio.  The way I estimate ratios is to run a small
aliquot of my purified fragments (i.e. after cutting, electrophoresis, and
gel purification) and stain with EtBr.  Intensity of staining is
proportional to the mass of the fragment, so you can get an estimate of
molar ratios from that.  For example:  If your insert is 1000 bp, and your
vector is 3000 bp, and the bands look equally intense on the gel, then
they have roughly the same mass and therefore there are 3 times as many
insert molecules as vector molecules.

>3. What is the correct composition of an
>selective blue white screening agar

If you're doing subcloning, and there's nothing tricky about it, then you
really shouldn't need blue-white selection.  95% of the colonies should
be correct.

Good luck.

Ian 

-- 
    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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