IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/25

From OpenWetWare
Jump to: navigation, search
Igem-logo-150px.png iGEM Project name 1 Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

dspB Track

  • O/N culture taken out at 1003
  • Transformants taken out at 1003
    • Results: 1,4,5,8,11,14,15,his,no-his: TMTC
    • Results: AA,TB: no colonies

Colony PCR

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1575
10mM dNTP0.5x157.5
sdH2O12.15x15182.25
Phusion polymerase0.1x151.5
MgCl22x1530
DMSO - 5%1.25x1518.75
10uM fw primer (G1004)2x1530
10uM re primer (G1005)2x1530
Total25375

PCR Tubes: 1,4,5,8,11,14,15,his,n1,n2,n3,n4,n5,W(H2O control)

  • Pick colony from respective plate and place in respective tubes

PCR Cycles:

  • 98C @ 3min
  • Cycle 27x:
    • 98C @ 10 sec
    • 72C @ 30 sec
    • 72C @ 40 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1148
End: 1251

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
1458111415100 bp ladderhisn1n2n3n4n5Wn2

Results:
File:100825 dspB 19.tif

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
100 bp ladder (quick load)1458111415100 bp ladder(iGEM)hisn1n2n3n4n5Wn2100 bp ladder (Quick load)

Results:
File:100825 dspB 20.tif

Restriction Digest Chlor backbone psb1C3

  • Protocol: biobrick (digesting)
Restriction Digest Supermix
REAGENTS1 RXN VOLUME (uL
Buffer 25
BSA0.5
ddH2O37
Total42.5
  • Add: 5uL of backbone + 5uL of insert
  • Enzymes: EcoRI and PstI (1uL each to each tube)

Biofilm Track

Eric F. + Melody

Biofilm Growth Protocol Day 3 (100823E + M)

  • 100823E removed from incubator @ 1100
    • Controls showed no growth (woot woot)
    • Followed procedure and placed in biosafet cabinet overnight
  • 100823M removed from incubator @ 1335
    • Growth in control wells C4, C6, D10
      • Some showed a distinct colony
    • Contamination in growth wells C9, E5, D9
      • Black dots - looked like growth of some other bacteria

Biofilm Growth Protocol Day 2 (100824E + M)

  • Removed O/N cultures from incubator @ 1100 (20 hrs growth)
  • Plate layout below:
100824E + M Plate Layout
Row 3 4 5 6 7 8 9 10
C C 8 C 8 R C R C
D 8 C 8 8 R C R C
E 8 8 C C C R C R
  • Note:
    • Controls chosen at random
    • R = RN4220
    • 8 = 8325-4
    • C = control
  • 100824M into incubator @ 1635
  • 100824E into incubator @ 1642
    • 100824E-E10 not fully filled, disregard all data from it