Paul.xu at 15:15, 11 July 2013

2013-07-11T15:15:47Z

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== Expression Vector Construction ==

A unique Sfi I site is inserted which after digestion with Sfi I create N-terminal Pfl MI compatible end and C-terminal BglI compatible end. See : 1783 pT7A31 

== Recursive directional ligation (concatemerization) ==

clone repetitive element into pEC. This is the parental vector 

digest donor (repetitive element) in pEC (parental vector) with PflM1 + BglI 

Digest destination vector (also parental vector if it is first round) with PflMI, dephosphorylate with CIP (1 unit of CIAP per pmole of vector for 1 hour at 50°C) and then purify using a  spin column. 

ligate and transform 

Screen by colony PCR and digestion 

Verify by sequencing 

== Subcloning ==

Subclone repetitive gene from final repetitive contruct in pEC into pT7A31 using 

donor: PflM1 + BglI 

pT7A31 vector: Sfi1, CIP treated vector 

Dephosphorylate the linearized vector (e.g., using 1 unit of CIAP per pmole of vector for 1 hour at 50°C). Heat-inactivate the CIAP for 10 minutes at 75°C. Gel purify the linearized, dephosphorylated vector 

Other vectors can be modified with one of these enzymes: AlwNI, BglI, BslI, BstAPI, DraIII, MwoI, PflMI, and SfiI, although it must be unique 

ligation and transformation 

 

== Verification ==

Short repetitive gene: PCR to identify positive colonies, then digestion and sequencing 

Long repetitive gene:  digestion and sequencing 

 

 

 

[[Category:Protocols]]

Synthesis of long repetitive genes - Revision history

Paul.xu at 15:15, 11 July 2013