ligation/transformation: HELP ME PLEASE!!

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Thu Mar 9 05:32:21 EST 1995


In article <lyons-0803951144010001 at cyclid.demon.co.uk>, lyons at cyclid.demon.co.uk (Alan Lyons) writes:
>In article <D52oz7.5n5 at news.cis.umn.edu>, shin at cis.umn.edu (Shin Enomoto) wrote:
>
>> HARDIES at THORIN.UTHSCSA.EDU wrote:
>> : Heidi Moss writes:
>> 
>> : > [having lots of trouble cloning some HindIII fragments]
>> : Could it be a sequence problem?? After all, these are supposedly 
>> : 5'/promoter clones that have an intron and exon present, based on initial 
>> : analysis. Could there be weird palindromes or repeats that the bacteria 
>> : doesn't like?? What part to you think is throwing me off?? Also, I just 
>> : did an experiment where I found that the gene is also slightly induced by 
>> : IFNB. I am suspecting a GAS element in the promoter, perhaps. It is a 
>> : far-fetched hunch, but could that cause a problem?? I am ignorant. I 
>> : don't know what to do now.
>> 
>> 
( I am sorry that my newsreader is unable to incorporate the entire thread, so
most of is deleted here )

It seems from looking back into this newsgroup into the articles posted by the
same person, that Heidi is doing something very similar to our project, that is
isolating a promoter region from a mammalian genomic library in lambda FIXII.
However, all this advice given to her may be irrelevant since there are no data
on how exactly the experiment is done. I would suggest a simple test before
any suggestion on the "poisoning sequences" is considered. This test comes
from an old article by J.Messing on M13 cloning.
1. Make up 4 ligation reactions with 20 ng of dephosphorilated vector in each,
and add: to No1 no insert, to No2 approximate amount to get 1:1 molar ratio,
to No3 3:1 and to No4 10:1
2. Repeat this with an unrelated insert or a digest with the same sticky ends
3.Add 0.1 Unit of ligase to each and incubate for 1-4 hours. Transform 1/3 of
each ligation mixture into a 50-100 ul competent cell aliqout, include also
0.1 ng of uncut vector as the 9th transformation.
4. Plate out 1/5 of each reaction onto 90 mm plate and count colonies.
5. The 9th plate should contain more then 500 colonies ( competent cell check )
In other plates, no insert plate should show a few colonies, on other plates
in this series the number of colonies should be increasing with the amount of
the insert added. If this number is decreasing, you've got the problem either
with the sticky ends integrity ( these days quite unlikely ) or with
"unclonable" sequences.
My point is that it is not very well known that some ends, particularly EcoRI,
HindIII and PstI, are ligated much faster than other (sticky) ends. Thus using
"1 microliter of ligase" ( =1-6 Weiss Units ) can easily screw things up, since
ligation at high ligase concentration produces chains and nets, instead of
transformable molecules. This is not an obvious problem, since as DNA
purification methods are improved, LESS ligase should be used than it used to
be ( e.g. ligation in agarose does require more ligase, than genecleaned DNA ).
Hope this helps

*************************************************************************
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



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