Melbourne/Plan:Gas vesicles

From 2007.igem.org

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#      Sequences:
#      Sequences:
##      Ncbi genebank AF053765  [[Melbourne/AF053765|(local copy)]]  [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] [[Melbourne/AF053765FASTA| (FASTA seq.)]]
##      Ncbi genebank AF053765  [[Melbourne/AF053765|(local copy)]]  [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] [[Melbourne/AF053765FASTA| (FASTA seq.)]]
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##      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)]
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###Total 2961 base pairs:
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###HindIII cuts at 719
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###EcoRI cuts at 707
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###PstI cuts at 701
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###Xbal cuts at 677
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###SpeI cuts at 683
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# Recovery of genes: (2 days)
# Recovery of genes: (2 days)
## Recover the plasmid from paper provided into solution.(method)
## Recover the plasmid from paper provided into solution.(method)

Revision as of 01:30, 10 July 2007

Steps:

  1. Sequences:
    1. Ncbi genebank AF053765 (local copy) (original source) (FASTA seq.)
    1. pBluescriptIIKS+ [1] [2] map) [3] | (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)
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