Re: [ccp4bb] Quikchange cloning: Insert length

- 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: Quikchange cloning: Insert length
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2008-12-01

Next message:
Subject: Re: Program to fill unitcell randomly
From: Ethan Merritt merritt {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2008-12-01

Subject: Re: Quikchange cloning: Insert length
From: Artem Evdokimov artem {- at -} XTALS {- dot -} ORG
Date: 2008-12-01

It helps to remember that PCR does have an upper limit of total
double-stranded DNA content (regardless of its molarity!) after which it
does not work any more (due to the competition of the polymerase for
non-specific dsDNA versus primer-substrate pairs).

Therefore the theoretical limits on this form of cloning are imposed by the
molarity of two DNA fragments, the fidelity/processivity of your PCR enzyme,
and the quality of the reaction mix. In practice it helps to also consider
the frequency of PCR errors as well as certain other factors.

I've done this sort of cloning with ~4 KB inserts, however the use of such
long inserts required optimization of conditions and was not 100% successful
(unlike the relatively simple insertion of 0.5-1.5 KB).

With long inserts it is *critical* to have highly purified and highly
homogenous starter DNA as well as (individually selectable) a correct
template/insert ratio.

Artem

-----Original Message-----
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji
Edayathumangalam
Sent: Monday, December 01, 2008 3:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Quikchange cloning: Insert length

Hi Folks,

Sorry for the non-xtallo posting.

I am curious to hear what is the longest insert anyone has cloned
using a modification of the Quikchange cloning strategy. Basically,
ligation-independent cloning by strapping on homologous regions of
the vector onto the primers which also generate the initial PCR
product. I plan to proceed with my insert which is ~ 2kb and am
curious to get some feedback if you have successfully cloned inserts
> 1.5kb using the above strategy.

Many thanks.
Raji

CCP4bb navigation

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

Previous message:
Subject: Re: Quikchange cloning: Insert length
From: Dima Klenchin klenchin {- at -} FACSTAFF {- dot -} WISC {- dot -} EDU
Date: 2008-12-01

Next message:
Subject: Re: Program to fill unitcell randomly
From: Ethan Merritt merritt {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2008-12-01