Team:Lethbridge/Notebook

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Revision as of 18:45, 9 September 2009



Igembanner.jpg




Calendar


May

May 5

Roxanne, Megan, Alix, Mackenzie, Sebastian

  • prepared Lb Media in test tubes -prepared 250mL LB agar for plates
  • Prepared 500nM Tris-HCl (pH 8.0), EDTA (pH 8.0, 500nm) TE Buffer (pH 8.0), GTE (Solution I), 2M NaOH, 10% SDS

May 6

Roxanne

  • Autoclaved LB test tubes, LB agar, Tris-HCl, EDTA, TE, GTE
  • Added ampicillin to LB agar (100ug/mL) -Poured LB + amp plates

May 12

Roxanne

  • Prepared LB agar
  • added Tetracyclin (100ug/mL) –
  • Poured LB + ampt plates

Roxanne, Alix, Ashley, Megan, Mackenzie, Kirsten

  • transferred pE43 (DH5α) cells from gylcerol stock to 5mL LB+Tet test tube (x8)
  • placed in orbital shaker overnight at 37°C

May 13

Roxanne

  • Removed test tubes from shaker (16hrs incubation) and placed in deli fridge

May 14

Roxanne, Alix, Ashley, Mackenzie, Kirsten

  • Preformed alkaline lysis miniprep on pE43 plasmid -lost plasmid from 2 tubes
  • obtained 6X pE43 plasmid (2X ohbR, 2x ohbB, ohbA, ohbC)

May 19

Roxanne, Alix, Ashley, Kirsten, Megan, Mackenzie

  • Gel Electrophoresis : 1% agarose in TAE

Lane 1: Ladder

Lane 2: ohbR1

Lane 3: ohbR2

Lane 4: ohbC

Lane 5: ohbB1

Lane 6: ohbB2

Lane 7:ohbA

Lane 8:ohbC

Lane 9: ohbB1

Lane 10:ohbB2

  • DNA quantification by UV spec 260nm : 2.013 280nm: 1.105 260/280 : 1.984 Concentration: 2517µg/mL PCR of ohbA, (ohbB)x2, ohbC, (ohbR)x2
  • Took picture of the gel - it did not turn out.

May 21

Roxanne

  • Made 50X TAE Buffer
  • Running PCR amplicons & RS/pSB1A2 ligation product on a 1% agarose gel
  • Jeff gave tutorial on primer design and quick-change mutagenesis to Kirsten, Ashley, Mackenzie, Fan, Lisza, Roxanne

May 26

Roxanne, Alix, Megan, Mackenzie, Kirsten

  • Gel did not turn out gave tutorial on open wetware and the igem registry

May 28

Roxanne

  • reran the gel from may 19th, only primer dimers present

Roxanne, Ashley, Alix, Kirsten, Mackenzie, Megan

  • Redoing the PCR of ohbA, ohbB, ohbC and OhbR with Phusion DNA Polymerase

Roxanne

  • refilled the tip boxes

May 29

Roxanne

  • prepared glycerol stocks for autoclaving


June

July

July 6th

Jeff, Mackenzie, Ashley

Using 1/10 and 1/100 dilutions of pTET and EYFP to perform large scale digests.

  • pTET:PstI and SpeI
  • EYFP:XbaI and PstI

4 reactions each:

  • single digest (PstI/SpeI and XbaI/PstI)
  • double digest
  • no digest

100µL each, means we need 400µL total for each.

1/10 dilution=40µL DNA, 360µL water

1/100 dilution=4µL DNA, 396µL water

Reaction set up:

  • 88µL DNA
  • 10µL buffer Tango (10x)
  • 1µL RE1 (or water)
  • 1µL RE2 (or water)
  • =100 µL total

16 reactions run overnight @ 37° C

Kirsten, Lisza

TetR Q04400-plate 1 well 16p-pSB2K3

LacI promoter-R0010-plate 1 well 1d-Amp

pBAD-I13453-plate 1, well 1n-pSB1A2

strong RBS-B0030-plate 1 well 1h-pSB1A2

med RBS-B0030-plate 1 well 2I-pSB1A2

10µL water into wells

Transformation:

2µL DNA (TetR, LacI, pBAD, med RBS, strong RBS) into 25µL competent DH5α cells (from the -80 fridge). Pipet up then down and swirl. One ice for 30min, then heat shock at 42° C for 45 sec. Ice for 1 min, add 250µL LB broth (from fridge) and incubate in shaker for 1 hour. Plate onto ampicillin plates (pBAD, LacI, strond and med RBS) and kanamycin plate (TetR) into incubator overnight. Control: 2.5µL water and 2.5µL DH5α.


August

September

October

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