>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 <[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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