IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/30

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psB Plasmids
Removed plates from incubator at 0840 Results: This confirms the psB1C3 backbone from last year does contain ccdB. This is useful information because we've been operating under the assumption of the presence of ccdB despite it being absent from this year's parts registry. Eric Finlay 12:21, 30 June 2010 (EDT)

Emily
Poured 12 kanamycin antibiotic plates. Last bottle of 400ml LB agar used. Need to make more LB

Phillip Chu
1. Checked the status of transformations. 5/6 plates had at least 2 colonies show up. Will leave plate 6 overnight to see if anything grows. Control plate had too many to count.

2. PCR'd 2 colonies from each plate to verify that they contain the parts desired (promoter+ RBS + fluorescence protein + terminator). Using Vf2 and Vr primers, the length should be approximately 1-1.1 kb.

3. Ran a gel of the PCR products using 110 V, 0.8% agarose, and 45 minutes run time.

4. Gel showed no bands. 100 bp ladders (flanking the PCR product lanes) also did not show up. Problem could be stain deactivation due to high temperature when pouring the gel (stain was added to the molten agarose). Will run the gel again with remaining PCR product tomorrow. Stain will be added with agarose is cooler.

Jason

 * Transfered 13mm glass tube of staph into cuvette + smaller glass tube (7mm?)
 * Streaked two plates of staph RN4220 in LB-only plates. Staph obtained from liquid overnight culture made on June 29th.
 * One sample extracted from cuvette, one sample obtained from (7mm?)glass-tube
 * Stored in 37C box at 3:30pm

Melody
Made 2 new glycerol stocks of staph (Staph obtained from liquid overnight culture prepared on June 29th. Taken out on June 30th)
 * Culture used = 250ul ; Glycerol volume = 750ul

Colony PCR

 * Refer to common protocols: "Colony PCR"
 * Colony PCR for individual colonies --> plates: 6T, 8T, 10T, 11T
 * 20 colonies for each plate, therefore 80 PCR samples in total
 * Split into 2 microcentrifuge tubes since it won't fit in 1 centrifuge tube:

Note: MME and MMF calculations include 80 rxns + 2 extra (for pipeting error) + H2O controls (3); original master mix in Table 1 calculations only contain 80 rxns.

PCR Cycles:
 * When picking colonies from the index plate, may have put two different colonies in 8T5 and 8T6.
 * 95C @ 15 min
 * 39 cycles:
 * 94C @ 30 sec
 * 56C @ 30 sec
 * 68C @ 2 min


 * 68C @ 20 min
 * 10C @ hold

Start time: 1013 End time: 1313 Placed in 4C fridge Vicki Ma 20:57, 1 July 2010 (EDT)