Re: [ccp4bb] regarding cloning

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

Previous message:
Subject: Re: regarding cloning
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2008-09-02

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From: Ron hudson hudson {- dot -} genome {- at -} GMAIL {- dot -} COM
Date: 2008-09-03

Subject: Re: regarding cloning
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2008-09-02

>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 attractive alternative:

6. "QuickChange cloning". Plasmid-, sequence- and restriction
enzyme-independent, only requires common PCR reagents. Clearly, it was
developed independently in several labs.

Basically, your PCR primers contain sequences that anneal to the plasmid.
Then all you do is set up QuickChange but use PCR fragment in place of
mutagenic primers.

Refs: Chen et al., 2001, Biotechniques, 28: 498-505; Miazaki and
Takenouchi, 2002, Biotechniques, 33:1033-1038; van den Ent and Lowe, 2006,
J Biochem Biophys Methods, 67:67-74.

We now do cloning this way. We use "PfuFusion Ultra II" polymerase because
it's so much better enzyme than anything else (better than "Phusion"). On
couple occasions and with a little more work, I was even able to get from
receiving primers on day1 to running a gel for expression/solubility test
on day3.

Dima

CCP4bb navigation

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

Previous message:
Subject: Re: regarding cloning
From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL
Date: 2008-09-02

Next message:
Subject: about pattersom map and self rotation function
From: Ron hudson hudson {- dot -} genome {- at -} GMAIL {- dot -} COM
Date: 2008-09-03