I want to draw attention to some of the early unruly response(s) to this question.

First of all, no one is forcing anyone to respond to each and every message that is posted on the CCP4 bulletin board.

If you are so keen on responding, please be respectful and considerate and think twice before you click on the send icon on your computer with derogatory remarks.


Thanks
Sridhar Prasad

-----Original Message-----
From: CCP4 bulletin board on behalf of Jan Schoepe
Sent: Sat 8/30/2008 8:52 PM
To: [log in to unmask]
Subject: [ccp4bb] AW: [ccp4bb] hi / cloning stuff
 
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 <[log in to unmask]> schrieb am Sa, 30.8.2008:
Von: vijay srivastava <[log in to unmask]>
Betreff: [ccp4bb] hi
An: [log in to unmask]
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
 




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