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)


 * Samples: 3 from plate 3 & 3 from plate 4 + 1 H2O + 1 extra = 8 samples


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

O/N culture for miniprep

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

Biofilm Growth cont.
We can now proceed with Day 2 of the Biofilm protocol. 8325-4 should be picked and incubated w/o shaking by 1500
 * control showed no growth
 * both RN4220 tubes (1 for Eric, 1 for Melody) did show growth
 * TSA plate of 8325-4 also showed growth

Plan
Test 1 Test 2
 * Make a 10% glucose stock (10g/100mL)
 * Clear out old/useless falcon tubes
 * Make 0.5, 1, 2, 4% glucose + TSB stocks
 * 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]
 * 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
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
Note:
 * C means control
 * 4 means TSB + 4% glucose w/ RN4220

Calculating Volumes Needed
Test 1 Test 2
 * 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
 * Multiply protocol by 10, so 300μL of culture and 30mL of TSB + 4% glucose

Making Glucose Stock
Below: Recipes for making the 4 different TSB + Glucose solutions
 * First make 10% solution - 800mL


 * 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