Team:Lethbridge/Notebook

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May

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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