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Team:Brown/Notebook/July27
From 2010.igem.org
Tuesday, July 27, 2010
Plans for inserting WillRS into pNoTat
- Digest pNoTat with NcoI and BamHI
- 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
