Melbourne/Plan:Gas vesicles

From 2007.igem.org

(Difference between revisions)
m (Melb:Plan:Gas vesicles moved to Melbourne/Plan:Gas vesicles)
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Steps:
Steps:
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# Recovery of genes:
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# Recovery of genes: (2 days)
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## Recover the plasmid from sample provided into solution.
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## Recover the plasmid from paper provided into solution.(method)
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## Transform E.Coli strain DH5alpha. Screen with Amp
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## Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
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## Culture->Establish Supply of Plasmid
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##     pick 3 colonies of each
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## Overnight Culture x9(6 above and 3 form agar block provided)
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##      Miniprep
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##      Produce glycerol stocks
## Confirm presence in recovered sample using digest.(HindIII)
## Confirm presence in recovered sample using digest.(HindIII)
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## Induce translation IPTG and confirm transcription RT-PRC, and Translation (buoyant phenotype).
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##      ->Established Supply of Plasmid
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# Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
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##      Confirm transcription RT-PRC,  
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##      Confirm translation (buoyant phenotype method).
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##      Confirm translation (Namarski optics (direct interferance contrast microscopy method).
# Removal of four biobrick like restriction sites all in GvpL.
# Removal of four biobrick like restriction sites all in GvpL.
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## SpeI [ACTAGT] (not present)
## SpeI [ACTAGT] (not present)
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# Insertion of biobrick required restriction sites by PCR primer modification.
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# Insertion of biobrick required restriction sites by PCR primer modification.(method)
## Design of primers
## Design of primers
## Primer generation
## Primer generation

Revision as of 00:01, 10 July 2007


Steps:

  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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