IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/24

From OpenWetWare

Jump to: navigation, search
iGEM Project name 1 Main project page
Previous entry      Next entry

Biofilm Track

Melody

Biofilm Protocol Day 1

  • Note: Each "batch" of O/N cultures include one test tube of control, RN4220, and 8325-4
    • Note: I used the timer/clock on the lab bench for the timing
  • Batch 1: in @ 4:03 p.m.
  • Batch 2: in @ 5:10 p.m.
  • Batch 3: in @ 6:06 p.m.
  • Batch 4: in @ 4:03 p.m.
  • Batch 5: in @ 4:03 p.m.
  • Batch 6: in @ 4:03 p.m.

dspB Track

  • 1/100 dilution of pre-dspB and Pcon in 37C shaking incubator at 12:10pm
  • O/N culture in 4C
  • 1/100 dilution of Pcon growing in 37C shaking (AMP not CHLOR) at 3:30pm
  • 1/100 dilution of:
    • pre-dspB OD600=1.054
    • P-con OD600=1.344

PCR using Rafael's method Attempt #5

  • Using new PNKed primers and Taq
PCR Master mix
Reagent1x rxn volume (uL)
10x Thermopol buffer2.5
10mM dNTP0.5
sdH2O13.65
Taq Polymerase0.1
MgCl22
DMSO - 5%1.25
gDNA1
fw rxn 22
re rxn 22
Total25
  • Tubes: rxn2, W

PCR Cycles:

  • 95C @ 2min
  • Cycle 30x:
    • 95C @ 30 sec
    • 58.5C @ 10 sec
    • 72C @ 80 sec
  • 72C @ 10 min
  • 10C @ hold

Start: 4:17pm
End: 5:48pm

Gel verification colony PCR products

  • Protocol: gel verification protocol in Protocol (SOP)
  • Machine conditions: 0.5x TBE buffer, 100V, 45min

Gel orientation:

Gel orientation
ABCDE100bp ladderFGHIW1 kb ladder

Results:

cPCR NCC colonies to verify before making glycerol stock

PCR Master mix
Reagent1x rxn volume (uL)
5x Reaction buffer5
10mM dNTP0.5
sdH2O12.15
Phusion Pol0.1
MgCl22
DMSO - 5%1.25
G10042
G10052
Total25
  • Tubes: NCC, Poco, Pcon, W
    • Poco and Pcon are for QS track

PCR Cycles:

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

Start: 7:26pm
End: 8:29pm

Gel verification colony PCR products

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

Gel orientation:

Gel orientation
NCCPoco100bp ladderPconW

Results:

O/N culture

  • O/N culture of NCC made with 1.5mL LB broth for glycerol stock
    • used 1.5uL of C1 antibiotic
    • put into 37C shaking incubator at 6:40pm


Personal tools