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.
Hint for the future: Dead glycerol stocks, even dead (say, a year old)
clone on a plate, still contain your plasmid. Do a regular miniprep with
it, transform - 99% of clones will contain your plasmid.
Dima
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: [email protected]
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.
<http://in.rd.yahoo.com/tagline_mail_2/*http://help.yahoo.com/l/in/yahoo/mail/yahoomail/tools/tools-08.html/>Get
it now
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