Hi, I think your question is quite reasonable no matter what others say about
it. There were even people asking about how to make buffers and then a big
discussion about the HH equation arose. And we should not forget that modern
crystallization starts with cloning ;-) Anyway, you should have given us some
more details about your experiments (cells, vectors etc.) and maybe a better
subject.
If you want to clone "something", it is a good idea to do it in silico (e.g.
with Clone Manager or Vector NTI) before you walk into a wet lab to check, that
things can work as desired or if there are fishy things like internal
restriction sides etc.
Let me tell you a story from my PhD thesis, maybe this will help you somehow. I
started with cloning 10 genes into a before modified pET vector (for E. coli
overexpression). After cleavage of the vector I got cohesive ends ("sticky
ends"). The genes were PCR amplified from the chromosome of another bacterium.
So things should have been very straight forward. Sticky ends should make sure
that the amplified genes were inserted in the right direction and the cleaved
vector should not ligate without the insert (and of course there was antibiotic
selection). But what I observed was often quite different from that. Often agar
plates stayed empty after transformation. That means, the cells were not able
to grow, because there was no vector in the cells that made them be able to
survive on the antibiotic plates. When I got colonies, the DNA gel looked very
strange (not possible to interpret) or the vector closed without the insert. In
this case, E. coli seems
to be able to "repair" the cohesive ends. Finally, I got it done but it took
me months. In one case, a plate seemed to be empty but I did not wanted to
throw it away so I let it stay a litte longer (~another day?, I do not remeber
exactly, but it was for a long time...) at 37° C than just over night (~0.5-1
days). Finally, one single colony showed up and it was one of the right ones.
With another vector it might be possible to do the same thing within weeks.
This is just cloning luck.
Another story, even if it could be embarassing to mention it here. In one case,
I got an Xtal structure but then I wanted to do some more activity tests. I
found out that my glycerol stocks were not alright any more and I had no
plasmid backups (do it!!!). So I had to clone, again. But in this case I used a
cloning kit from Qiagen (I have no commercial interests here). I got the insert
into the cloning vector from Qiagen very easily. Then I was able to amplify it
as much as I wanted and the ligation procedure was not so dependent from the
yield of the PCR reaction (high molar access of insert compared to the cleaved
vector) . Also in this case, cloning into the expresson vector was not
completely trivial (maybe 1-1.5 months) but much easier than without this kit.
So, it might be an idea to use a kit that you can amplify your insert as much
as you want. I know that these kits a pricy but your time is also expensive and
you also use/waste a lot of
chemicals for unsuccessful trials.
After I finished my thesis, another PhD student started to work with these
vectors and tried it for more than one year and then gave it up! So this vector
really seems to be a tough one. Thats why if nothing works, you might take
another vector into consideration. But before that you should check buffers,
ligase, restriction enzymes etc. and do in silico cloning. Another idea would
be do to de/phosphorylation (see textbooks).
Good luck!
Jan
--- vijay srivastava <[EMAIL PROTECTED]> schrieb am Sa, 30.8.2008:
Von: vijay srivastava <[EMAIL PROTECTED]>
Betreff: [ccp4bb] hi
An: CCP4BB@JISCMAIL.AC.UK
Datum: Samstag, 30. August 2008, 13:04
hi
i am facing problem in cloning,getting my insert and vector at the
correct position after digestion but after ligation colony is not
coming and if how it is coming than i am not getting my insert
Unlimited freedom, unlimited storage. Get it now
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