IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/12

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dspB Track

Re-do colony PCR on samples (transformants) from 100810

  • Protocol: common protocol (SOP)
PCR Master mix
Reagent1x rxn volume (uL)Master Mix
10x rxn buffer2.5x820
10uM FW primer2x816
10uM RE primer2x816
10mM dNTP1x88
sdH2O14.05x8112.4
Taq polymerase0.2x81.6
DMSO1.25x810
MgCl22x816
Total25200
  • Samples: 3 from plate 3 & 3 from plate 4 + 1 H2O + 1 extra = 8 samples
PCR Tubes
From plate 3:3T13T23T3
From plate 4:4T14T24T3
W (H2O control)
  • Colonies picked from index plate

PCR Cycles:

  • 95C @ 10min
  • Cycle 25x:
    • 94C @ 30 sec
    • 56C @ 30 sec
    • 68C @ 2 min
    • 68C @ 20 min
  • 10C @ hold

Start: 1134
End: 1324

Gel verification for 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
3T13T23T31kb ladder4T14T24T3W (control)

Results:
File:100812 DspB 15.tif

O/N culture for miniprep

  • 3T1 and 4T3 colonies taken from index plate were put into tubes. Tubes put into 37C shaker @ 1607

Biofilm Track

Eric F.

Biofilm Growth cont.

  • control showed no growth
  • both RN4220 tubes (1 for Eric, 1 for Melody) did show growth
  • TSA plate of 8325-4 also showed growth

We can now proceed with Day 2 of the Biofilm protocol. 8325-4 should be picked and incubated w/o shaking by 1500

Plan
  • Make a 10% glucose stock (10g/100mL)
  • Clear out old/useless falcon tubes
  • Make 0.5, 1, 2, 4% glucose + TSB stocks

Test 1

  • Testing effect of additional glucose on Biofilm growth (0.5, 1, 2 and 4% added)
  • For better data instead of doing experiments in triplicate (3x) we are doing them 6x
  • There will be 8 controls, 2 for each glucose concentration]

Test 2

  • Testing effect of location on plate on biofilm growth
  • 96 wells = 88 TSB + 4% glucose + RN4220 and 8 growth control wells
Test 1 96 Well Plate Layout
Columns
Rows 1 2 3 4 5 6 7 8 9 10 11 12
A X X X X X X X X X X X X
B X X X X X X X X X X X X
C X X C 0.5 0.5 0.5 0.5 0.5 0.5 C X X
D X X 1 1 C 1 C 1 1 1 X X
E X X 2 2 C 2 C 2 2 2 X X
F X X C 4 4 4 4 4 4 C X X
G X X X X X X X X X X X X
H X X X X X X X X X X X X

Note:

  • X means empty
  • C means Control
  • 0.5 means TSB + 0.5% glucose w/ RN4220
  • 1 means TSB + 1% glucose w/ RN4220
  • 2 means TSB + 2% glucose w/ RN4220
  • 3 means TSB + 4% glucose w/ RN4220
Test 2 96 Well Plate Layout
Columns
Rows 1 2 3 4 5 6 7 8 9 10 11 12
A 4 4 4 4 4 4 4 4 4 4 4 4
B 4 C 4 4 4 4 4 4 4 4 C 4
C 4 4 4 4 C 4 C 4 4 4 4 4
D 4 4 4 4 4 4 4 4 4 4 4 4
E 4 4 4 4 C 4 C 4 4 4 4 4
F 4 4 4 4 4 4 4 4 4 4 4 4
G 4 C 4 4 4 4 4 4 4 4 C 4
H 4 4 4 4 4 4 4 4 4 4 4 4

Note:

  • C means control
  • 4 means TSB + 4% glucose w/ RN4220
Calculating Volumes Needed

Test 1

  • Use number from protocol: 30μL diluted by 1:100 - so 3mL of TSB + x % glucose
  • 4 different %s of glucose therefore only need 120μL of culture

Test 2

  • Multiply protocol by 10, so 300μL of culture and 30mL of TSB + 4% glucose
Making Glucose Stock
  • First make 10% solution - 800mL

Below: Recipes for making the 4 different TSB + Glucose solutions

0.5% 1% 2% 4%
Total Volume 25mL 25mL 25mL 40mL
Volume TSB 23.75mL 22.5mL 20mL 24mL
Volume 10% Glucose 1.25mL 2.5mL 5mL 16mL
  • Followed protocol for Day 2 exactly
    • Exception: Test#2 Step 1: Diluted 300μL of culture by 1:100
  • Test 1 into incubator @ 1437
  • Test 2 into incubator @ 1452

8325-4 Biofilm Growth

  • Picked a colony from TSA plate, incubate w/o shaking in 5mL of TSB @ 37°C
    • In incubator @ 1630
    • 1 control w/ 4.5mL TSB was used