Team:Brown/Notebook/July27

From 2010.igem.org

Tuesday, July 27, 2010

Plans for inserting WillRS into pNoTat

  1. Digest pNoTat with NcoI and BamHI
  2. Ligate (overnight 4C or overnight 16C in PCR) pNoTat and WillRS

1) Double digest

20ul total volume

  • 7.8ul dH2O
  • 2ul 10X NEB Buffer3
  • 8ul DNA
  • 1ul NcoI
  • 1ul BamHI
  • 0.2ul BSA

1 hour at 37C. Started at 11:37AM.

PCR Cleanup (Qiagen Kit) of pNoTat digest

5 volumes of Buffer PB1 to one volume digest sample and mix. Centrifuge. Add 0.75ml buffer PE

2) Ligation of 4C overnight

15ul total volume

  • 7.5ul ligase buffer
  • 1.0ul T4 DNA ligase
  • 0.5ul vector (pNoTat digested and purified)
  • 1.5ul insert (WillRS)
  • 4.5ul dH2O

Double Digest of pAraC (R0080) and RBS-ChFP_TerTer (J06702)

Purpose - to join pAraC with ChFP to create a reporter construct

  • Digest pARaC w/ EcoRI, SpeI in buffer II (NEB)
  • Digest J06702 w/ EcoRI, XbaI in buffer II (NEB)

pAraC will be taken out of plasmid, whereas J06702 plasmid will be opened up

20ul total volume

  • 7.8ul dH2O
  • 2ul 10X NEB Buffer3
  • 8ul DNA
  • 1ul NcoI
  • 1ul BamHI
  • 0.2ul BSA

incubate 1hr@37C, starting at 3:20PM

PCR cleanup (using gel extract kit)

Ligation overnight @4C

  • 7.5ul ligase buffer
  • 1ul T4 ligase
  • 4.5ul dH2O
  • 0.5ul J06702 (vector)
  • 1.5ul pAraC (insert)

2 tubes (trials)

Testing M9 minimal media

  • 4mL M9min w/ XL1s
  • 4mL m9min not inoculated

Incubated @37C at 6:35PM. Also took 5ml min media w/ lovtap and lovtap2 and it grew overnight to saturation

Transformations of parts from registry

Used cells labeled XL1-B 7/16. We have recently discovered that these cells are contaminated with Kan resistance, so cannot yet transform Mnt because its plasmid uses Kan for selection.

  • RBS34 - 2010 plate 1 well 2M - AmpR
  • CI - 2010 plate 1 well 4E - AmpR
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