dspB Track
Re-do colony PCR on samples (transformants) from 100810
- Protocol: common protocol (SOP)
PCR Master mix
Reagent | 1x rxn volume (uL) | Master Mix |
10x rxn buffer | 2.5 | x8 | 20 |
10uM FW primer | 2 | x8 | 16 |
10uM RE primer | 2 | x8 | 16 |
10mM dNTP | 1 | x8 | 8 |
sdH2O | 14.05 | x8 | 112.4 |
Taq polymerase | 0.2 | x8 | 1.6 |
DMSO | 1.25 | x8 | 10 |
MgCl2 | 2 | x8 | 16 |
Total | 25 | | 200 |
- Samples: 3 from plate 3 & 3 from plate 4 + 1 H2O + 1 extra = 8 samples
PCR Tubes
From plate 3: | 3T1 | 3T2 | 3T3 |
From plate 4: | 4T1 | 4T2 | 4T3 |
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
3T1 | 3T2 | 3T3 | 1kb ladder | 4T1 | 4T2 | 4T3 | W (control) |
Results:
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
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