Alternative to cloning PCR-fragment directly into ve

Trond Erik Vee Aune trondaun at chembio.ntnu.no
Thu Jun 13 05:47:15 EST 2002


Dear Wolfgang,

Thanks for your helpful reply. I've given some further information and 
comments below.


> Since the number of cycles is low, also Taq should do the job. (You're
> interested in mutants anyway, aren't you :?)


Hehe, yes, but not too many in the screening vector ;)...If everything 
is okay I already have the mutations I need in the mutated fragment.


> If annealing doesn't work, introduce a third step in the PCR and use say 
> 30 sec or so for this. If you're lazy, you might set up a touch down 
> protocol and supply sufficient vector (say 0.5 ug, this would ensure in 
> theory that your "primers" are incorporated completely as
> soon as they bind).


Yes, I think it would be a good idea to use a lot of my vector to ensure 
  that all of my "primers" (the complete library) is hybridized. This 
way I minimize the loss.


> But actually, the "orthodox way" should
> work nevertheless!
> 
> Can you make sure that
> 
> 1) ligation itself is working (you also might try various vec/insert 
> ratios)


I've trying different ratios and have confirmed that the 5:1 molar 
(insert:vector) is optimal.

There is nothing wrong with the ligase nor the buffer.


> 2) transformation is effective (you need some highly comepetent cells like
> XL10 or homemade equivalents)


We have pretty good supercompetent DH5alpha which we make ourselves, 
I've also tried with some ultracompetent XL-10 from Stratagene.


> 3) you don't loose your material during
> purification - try to avoid as many potentially unneccesary steps as 
> possible
> (e.g. use DpnI and EtOH prec in your initial PCR instead of gel 
> purification
> etc. etc.).


After error-prone PCR I purify the reaction mix (50 microl.) on gel to 
get rid of template DNA, possible wrong fragments (none as far as I can 
see) and different components from the reaction (polymerase, dNTP and so 
on). Then I purify the fragments with Qiagen's QIAquik to get rid of 
agarose, EtBr and the TAE.

Then I cut the fragments in a double digest (with AgeI and SacI) and 
separate the molecules on another agarose gel. I end up with 30 microl. 
of purified DNA fragments with a concentration of about 40 ng/microl 
(estimated from gel). That gives a total amount of 2 micrograms.

It is clear that I lose a lot of my fragments during these two 
purifications, but I think that 2 micrograms should be sufficient to get 
the number of different clones I want.


> 4) you take appropriate means to reduce background (SAP, two different
> restriction enzymes, a third RE that cuts the vector inbetween the other 
> two
> (In this case I tend to be paranoid).


I always try to ligate only vector (negative control) in each cloning 
procedure. Tha amount of re-ligated vector has been nothing to worry about.


> 5) include a positive control where you
> clone a pure DNA fragment obtained by a RE
> digest.


I haven't done this yet. I will include this test next time.


> Do you have any clues where you might loose your materal?


I think my problem is that my RE (AgeI specifically) doesn't cut the 
inserts optimally. If it did not cut the vector optimally as well I 
should get a lot of re-ligated vector on my negative control.

I have just received an isoscizomer of AgeI called BshTI which should be 
better than AgeI. I'll try this tomorrow next to AgeI to compare the 
efficiences of these two REs.


> 
> How many individual clones do you need and how many colonies did you get?


Preferably more than 10k, at least 3k. My results are estimated to be 
less than 1k.

Regards,
Trond Erik





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