IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/31

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Biofilm Track

Melody

  • Took out O/N cultures of RN4220 and 8325-4 from non-shaking incubator @ 11:52 a.m.

Biofilm Protocol Day 2

  • Innoculated plates 100830M120+100830M222

100830M1 + 100830M2 96-well plate Layout

Row 3 4 5 6 7 8 9 10
C R R C R 8 8 8 C
D R R R R C 8 8 8
E C R R C 8 8 8 C
  • Note:
    • Controls chosen at random
    • R = RN4220
    • 8 = 8325-4
    • C = control
  • Incubated @ 2:00 p.m. w/o shaking

Overnight cultures

  • 2 O/N cultures of RN4220 and 8325-4
    • one in incubator w/ shaking
    • one in incubator w/o shaking
  • Both in @ 3:57 p.m.

DspB Track

PCR off gDNA for more PCR products

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1050
10mM dNTP0.5x105
sdH2O7.15x1071.5
Phusion polymerase0.1x101
MgCl22x1020
DMSO - 5%1.25x1012.5
Total16160
PCR Tubes
TubeContent
His1,His2,His3,His4dspB his
no his1,no his2,no his3,no his4dspB no-his
W (H2O control)
  • To His1,His2,His3,His4: add 2uL of up-his & dw primers each
  • To no his1,no his2,no his3,no his4: add 2uL of up & dw primers each
  • To all, add 5uL of DNA

PCR Cycles:

  • 98C @ 30s
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1234
End: 1335

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Changes: 1% agarose gel
  • Machine conditions: 0.5x TBE buffer, 100V, 60min

Gel orientation:

Gel orientation
his1his2his3his4100bp ladderno his1no his2no his3no his4W (control)

Results:
File:100831 dspB 22.tif

PCR purification

  • All 4 tubes of His in 1 spin column (88uL + 440uL PBI)
  • All 4 tubes of no his in 1 spin column (88uL + 440uL PBI)
  • [His] = 126.3ng/uL
  • [No his] = 196.8ng/uL