1. Typical [restriction based] sub-cloning
2. Go through (TOPO)-TA cloning
3. Gateway cloning
4. LIC, Ligation independent cloning
5. SLIC, Sequence and Ligation independent Cloning.
At the risk of being repetitive (since I posted it in a previous "cloning"
thread), there is another very attrac
tin board [mailto:[EMAIL PROTECTED] 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
(I was managing to insert
multiple copies), and it still worked: with only one copy of the insert
inserted.
Zhijie
- Original Message -
From: vijay srivastava
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, September 01, 2008 3:05 AM
Subject: [ccp4bb] regarding cloning
tor.
>
>
>
> 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:[EMAIL PROTECTED] *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
-
Date: 1-sep-2008 03:06:29 -0400
From: "vijay srivastava" <[EMAIL PROTECTED]>
Reply-To: <[EMAIL PROTECTED]>
To:
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 enz
T/A cloning utilizes the overhangs left by certain polymerases as cloning
handles. To lower background, the vectors for TA cloning are often designed
to contain a rare cutter which is used during ligation to constantly re-cut
the self-ligated vector, so this way only the insert ligation product is
CCP4 bulletin board [mailto:[EMAIL PROTECTED] 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 u
d then things have worked like a charm!
Hope that helps.
Raji
-Included Message--
>Date: 1-sep-2008 03:06:29 -0400
>From: "vijay srivastava" <[EMAIL PROTECTED]>
>Reply-To: <[EMAIL PROTECTED]>
>To:
>Subject: [ccp4bb] regarding cloning
>
>H
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 digesti
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