From 2007.igem.org

Steps:

1. Sequences:
   1. Ncbi genebank AF053765 (local copy) [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] (FASTA seq.)

1. 1. pBluescriptIIKS+ [http://www.stratagene.com/products/displayProduct.aspx?pid=267|(stratagene)] [http://www.stratagene.com/vectors/sequences/pbl2ksp_s.txt|(sequence)] [http://www.stratagene.com/vectors/restriction_sites/pbl2ksp_r.txt|(restriction map)] [http://www.stratagene.com/vectors/maps/pdf/pBluescript%20II%20KS+_%20webpg.pdf|(map)] [http://www.stratagene.com/manuals/212205.pdf | (Manual)]
      1. Total 2961 base pairs:
      2. HindIII cuts at 719
      3. EcoRI cuts at 707
      4. PstI cuts at 701
      5. Xbal cuts at 677
      6. SpeI cuts at 683

1. Recovery of genes: (2 days)
   1. Recover the plasmid from paper provided into solution.(method)
   2. Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
   3. pick 3 colonies of each
   4. Overnight Culture x9(6 above and 3 form agar block provided)
   5. Miniprep
   6. Produce glycerol stocks
   7. Confirm presence in recovered sample using digest.(HindIII)
   8. ->Established Supply of Plasmid

1. Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
   1. Confirm transcription RT-PRC,
   2. Confirm translation (buoyant phenotype method).
   3. Confirm translation (Namarski optics (direct interferance contrast microscopy method).

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.(method)
   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)