Re: [ccp4bb] regarding cloning

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

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

Previous message:
Subject: Re: regarding cloning
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2008-09-01

Next message:
Subject: Como Crystallography School registration deadline extended for the last time!
From: Derek Logan Derek {- dot -} Logan {- at -} MBFYS {- dot -} LU {- dot -} SE
Date: 2008-09-01

Subject: Re: regarding cloning
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-09-01

Hi,

First of all - I am curious why did you decide to put in an extra step (the
T/A cloning into an intermediate vector)? You can happily digest your PCR
product with NheI/BamHI, clean up and ligate into the appropriately digested
pET-23a(+). If you have issues, you should definitely try this.

Now, since you do have an intermediate step - did you verify that everything
was OK after havig subcloned your insert into whatever vector you're using?
Did you sequence the insert and most importantly did the sequencing confirm
the nature of the linker regions?

The enzyme pair that you chose has a slight issue with digestion buffer -
most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where
Bam still has '100% activity' - however, in buffer 2 you can have star
activity of the Bam due to the somewhat lower salt concentration (50 mM
instead of the optimum 100 mM). It's not impossible to imagine that you have
issues with digestion. This can be easily avoided by sequential digestion
although of course it's slightly more work (but if you cut out the T/A
cloning step that's actually still faster).

So, in conclusion the most likely issue is digesiton (probably of the pET
vector, to be more specific). Next likely issue could be ligation - make
sure that you base your ligation ratio on the gel intensity of the bands as
well as on the OD260 of your DNA. Faulty primers are not likely to be an
issue since you seem to be able to restrict your insert out of the
intermediate vector.

Please note that you can often use SpeI or XbaI instead of Nhe since they
have compatible sticky ends. Clearly this depends on the vector you're
working with and I am too lazy to look up pET23 polylinker.

Artem

_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of vijay
srivastava
Sent: Monday, September 01, 2008 3:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] regarding cloning

Hi,

I am trying to clone a 1.2kb insert into a expression vector pET 23a through
T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in
the forward and reverse primer recpectively. I was succesful in subcloning
(T/A vector) and getting my insert at 1.2kb after 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 how
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.

_____

Be the first one to try the new Messenger 9 Beta! Click
n/> here.

CCP4bb navigation

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

Previous message:
Subject: Re: regarding cloning
From: Raji Edayathumangalam redayath {- at -} MAIL {- dot -} ROCKEFELLER {- dot -} EDU
Date: 2008-09-01