Ligation problems

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Sun Mar 19 13:03:55 EST 1995


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.
>
Perhaps I should have written "cloning" rather than "ligation". At one time
I was doing a lot of blunt-end ligations, using the same vector prep. When
you transform  1 ng of an uncut pUC into the cells I usually had, I got more
than 2x10E7 colonies per ug. If the same amount of vector was transformed after
it had been cut with EcoRI,
heat treated and religated, the number would drop by a factor of 10. If I did
the same with SmaI, the number would drop by a factor of 10E3. With the vector
which was dephosphorilated and ligated to an insert, this number ( dependent 
on the amount of insert added) would drop further ( but not to zero ). It could
be that my Sma was not the best, but if I relied on these numbers, I always got
the clones. The amount of vector and the range of insert amount which has to be
tried, is also coming from practice: a more theoretical insight would have
taken into consideration the actual length of both vector and insert, and had
to be in molar amounts, rather than in weight units. For that, see a more
general advice of Steve Hardies some two days earlier. 

>Dept. of Genetics
>U. of Naples
>Italy
>  
*************************************************************************
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
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