Thursday, August 12, 2004 , 20.40

Cloning of concatamers into pET AvaI

The AvaI concatamers are subcloned into a dephosphorylated pET AvaI expression vector by bulk ligation. The ligation producrs are transformed into SURE2 (recB-A, recJ-A) supercompetent cells (Stratagene) and the colonies analyzed directly by DNA purification and restriction digestion or by PCR colony screening using Platinum Taq DNA polymerase (Gibco-BRL). This is the most critical and tedious step since bulk ligation is used. We typically screen 50-300 clones to get a concatamer of eight or more monomers.



4 Comments »

  1. conoscevo un tizio che si faceva chiamare polimerase

    Comment by onq — 12 August , 2004 @ 21:01
  2. era troppo fatto e s’è perso i primer, o forse, sarà stato il bersaglio sbiadito, si sa che a 93° i colori se ne vanno. Non ci sono più le casalinghe di una volta.

    Comment by Sol7 — 13 August , 2004 @ 07:20
  3. il dr. Molese approva e consiglia questo blog.

    Comment by MarioMolese — 13 August , 2004 @ 10:05
  4. e che palle mi scoccio di tradurre

    Comment by ColonnelloLobanovski — 16 August , 2004 @ 03:04

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