IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/07/05

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BioFilm Track

  • Grew 2 sub-cultres in 10ml of staph + B2 (3ml) for OD curve and staph competent cell. Cell obtained

from glycerol stocks

  • Placed in AMBL Rotator (32C, 180rpm)
  • Start: 3:30pm (July 05,10)
  • End: 9:30am ( July 06, 10)

Misc

  • Poured Amp plates (18 plates)
  • Placed 2 ON E.coli competent cells in AMBEl rotator (32C, 180rpm)
  • Autoclaved 500ml LB Broth media
  • Autoclaved 600ml B2 media

dspB Track

Marianne and Vicki

Colony PCR

  • Protocol: Used 'colony PCR' in common protocols
  • Colony PCR for individual colonies:
  • 11T,12T
  • 5 colonies from each plate; therefore, 10 PCRs in total
Table 1. Master Mix
REAGENT1 RXN VOLUME (uL)MASTER MIX (uL)
10x rxn buffer2.5x1332.5
10uM FW primer (G1004)2x1326
10uM RE primer (G1005)2x1326
10mM dNTP3x1339
sdH2O15.3x13198.9
Taq polymerase0.2x132.6
Total25235
Table 2. PCR tubes
11T111T211T311T411T5H2O control (W)
12T112T212T312T412T5
  • For each respective tube, picked colonies numbered 3,5,7,9,11 in order and added to each microcentrifuge tube in ascending order. Ex. Colony #3 from plate 11T into 11T1.

PCR Cycles:

  • 95C @ 15min
  • 26 cycles:
  • 94C @ 30 sec
  • 56C @ 30 sec
  • 68C @ 2 min
  • 68C @ 20 min
  • 10C @ hold
  • Start PCR: 1351
  • End PCR: 1603

Gel verification

  • Protocol: gel verification protocol in iGEM training manual
Table 3. Gel orientation
layer 111T111T211T311T411T512T112T212T312T412T5100bp ladderW10T110T210T310T410T510T610T710T8
layer 210T910T101-T1110T1210T1310T14100bp ladder10T1510T1610T1710T1810T19
  • Machine conditions: 110V, 40 minutes, 0.5X TBE buffer
  • DNA: 10uL, loading dye: 2uL

Gel results:
File:100705 dspB 7.tif

Overnight culture

  • Protocol: From iGEM common protocols
  • Colonies: 8T3, 6T10, 6T11, 6T14
  • Cultures (tubes): 8T3, 6T10, 6T11, 6T14, control (LB broth+chlor)
  • Put cultures in 37C turntable in AMBL lab @ 1800

Vicki Ma 18:34, 6 July 2010 (EDT)