Ligation problems

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Mon Mar 20 08:27:47 EST 1995


In article <3khrmb$o8p at elna.ethz.ch>, bckraev at wawona ("Alexander Kraev" bckraev at aeolus.ethz.ch) writes:
>In article <3kf65g$4sb at sun01.iigb.na.cnr.it>, John and Alex <pulitzer at plauto.csata.it> writes:
>>bckraev at wawona ("Alexander Kraev" bckraev at aeolus.ethz.ch) wrote:
>>>
>>
>>> >
>>> >There is no real special technique, you just have to accept that it
>>> is not going to be as efficient as any sticky end-ligation. All you need
>>> for success is: phosphatased, gel purified vector; a lot of ligase ( 1-5
>>> Weiss units in 10 ul reaction mixture) and an excess of insert. Typically,
>>> 20 ng of vector and 20-100 ng of an insert are ligated in 10 ul. With
>>> average competent cells ( 10E7 colonies per ug ) this reaction should produce 
>>> a total of 200 colonies, of which at least 30% are recombinant. These
>>> figures in fact show that such a ligation is about 10E4 times less efficient
>>> than an average sticky-end one. Hope this helps, 
>>> 
>>Thats an impressive calculation! Does it take into account efficiency of  
>>circularization of the 3kb insert? Anyway I wish I knew how to do it.
>>Could you let us have some reference or hint on how you calculated the
>>10E4 factor difference? Thanks.
>>
Sorry, there is a mistake here, it must be "10E2 less efficient". And this is
not entirely a "calculation". The idea of this estimate is that if you have
cells with which you can get 10E7 colonies from 1 ug of uncut vector, you
would like to know how much this efficiency would drop if you supply the same
amount of vector, which was cut, phosphatased and ligated to an insert. You
certainly can not get over 10E7, right? So, from practice I knew that with
sticky end cloning I could get almost 10E6 per ug, although actually 20 ng were
transformed. Now, with blunt end cloning I found that this number is around
10E4 per ug  with the same vector and cells, which gives us 200 colonies
from 20 ng. Why this number was so low in my case, is another matter. But
for the experiment such as the one described in the original post, it is more
than adequate. Cheers,

*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



More information about the Methods mailing list