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Revision as of 22:04, 3 July 2007

Steps:

1. Recovery of genes:
   1. Recover the plasmid from sample provided into solution.
   2. Transform E.Coli strain DH5alpha. Screen with Amp
   3. Culture->Establish Supply of Plasmid
   4. Confirm presence in recovered sample using digest.(HindIII)
   5. Induce translation IPTG and confirm transcription RT-PRC, and Translation (buoyant phenotype).

1. Removal of four biobrick like restriction sites all in GvpL.
   1. EcoRI [GAATTC] in gvpL (2858)
   2. PstI [CTGCAG] in gvpL x 3 (2522,2900,3005)
   3. XbaI [TCTAGA] (not present)
   4. SpeI [ACTAGT] (not present)

1. Insertion of biobrick required restriction sites by PCR primer modification.
   1. Design of primers
   2. Primer generation
   3. Plasmid extraction from culture
   4. PCR
   5. Restriction EcoR1 & Spe1
   6. Gel separation
   7. Restriction of standard Library death plasmid EcoR1,Spe1.
   8. Ligation.
   9. Transform host with regulated POPS output
   10. Confirm dna, rna , protein (as for A)