From 2007.igem.org

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Revision as of 01:57, 9 July 2007

Steps:

  1. Recovery of genes SopII-HtrII:
        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)