IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/26: Difference between revisions

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*minipreps of v0120sense+pdk/pal intron parts
*minipreps of v0120sense+pdk/pal intron parts
*Digest vectors w/ Spe and Pst1 and ligate with antisense parts (already digested and gel purified)
*Digest vectors w/ Spe and Pst1 and ligate with antisense parts (already digested and gel purified)
**Result: [[Image:2-assembly_digest.jpg|50px]]
*Sequence review: clarified which parts of the sequences on the team page corresponded to sense/antisense.
*Sequence review: clarified which parts of the sequences on the team page corresponded to sense/antisense.


Revision as of 14:50, 26 July 2010

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Team Fence

PFX polymerase PCR

  • 1μL 10mM MgSO4
  • 2μL 10X pfx amplification buffer
  • 2μL 10X PCRx enhancer solution
  • 1μL F primer
  • 1μL R primer
  • 0.5μL pfx polymerase
  • 2μL DNTP

9.5μL total

DNA: 21ng/μL

  • 50 to 200μL DNA per reaction
  • 5 reactions
  • 4μL per reaction = 20μL sample total (MM)

9.5μL + 4μL = 13.5μL therefore 6.5μL H20 per reaction

MM

  • 5μL 10nM MgSO4
  • 10μL 10X pfx amplification buffer
  • 10μL 10X PCRx enhancer solution
  • 5μL F primer
  • 5μL R primer
  • 2.5μL pfx polymerase
  • 10μL DNTP
  • 20μL genomic DNA
  • 32.5μL H20

total volume:100μL

Arabidopsis PCR

Team Allergy

  • minipreps of v0120sense+pdk/pal intron parts
  • Digest vectors w/ Spe and Pst1 and ligate with antisense parts (already digested and gel purified)
    • Result:
  • Sequence review: clarified which parts of the sequences on the team page corresponded to sense/antisense.

Team Flavor

  • Digested miniprepped Mira/Brazz+StrepII+STOP constructs with Not1/Spe1 for ligation confirmation: failed
  • Ran PCR product of Valencia Orange genome to confirm: failed

  • Digested Mira/Brazz+StrepII constructs EcoRI/SpeI, STOP+V0120 EcoRI/XbaI
   Digestion Reactions
    Mira N:  888ng
    Mira C:  888ng
    Brazz N: 993ng
    Brazz C: 1032ng
    STOP:    781ng x 2 (max DNA available)



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