[ccp4bb] regarding cloning

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: SUMMARY List of conserved waters
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-01

Next message:
Subject: Re: SUMMARY List of conserved waters
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-01

Subject: regarding cloning
From: vijay srivastava vijaytechno {- at -} YAHOO {- dot -} CO {- dot -} IN
Date: 2008-09-01

Hi,
I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.Â The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesfulÂ in subcloning (T/A vector) and getting my insert at 1.2kb afterÂ double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some howÂ when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there.

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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: SUMMARY List of conserved waters
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-01

Next message:
Subject: Re: SUMMARY List of conserved waters
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2008-09-01