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

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Misc. Track

Eric F.

psB Plasmids

Removed plates from incubator at 0840 Results:

Plate Results
B2A Growth (TMTC)
B1B Growth(TMTC)
α1A Nothing
α2B Nothing

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

QS Track

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.

Biofilm Track

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

dspB Track

Marianne and Vicki

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
Table 1. PCR master mix ingredients
REAGENTS1 RXN VOLUME (uL)MASTER MIX(uL)
10x rxn buffer2.5x80200
10uM FW primer (G1004)2x80160
10uM RE primer (G1005)2x80160
10mM dNTP4x80320
sdH2O14.5x801144
Taq Polymerase0.2x8016
Total252000
  • Split into 2 microcentrifuge tubes since it won't fit in 1 centrifuge tube:
Table 2. Master Mix II
REAGENTMaster Mix E (MME)(uL)Master Mix F(MMF)(uL)
10x rxn buffer106.25106.25
10uM FW primer (G1004)8585
10uM RE primer (G1005)8585
10mM dNTP170170
sdH2O607.75607.75
Taq polymerase8.58.5
Total1062.501062.50

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.

Table 3. PCR Tubes/Samples
6T16T26T36T46T56T66T76T86T96T10
6T116T126T136T146T156T166T176T186T196T20
8T18T28T38T48T58T68T78T88T98T10
8T118T128T138T148T158T168T178T188T198T20
10T110T210T310T410T510T610T710T810T910T10
10T1110T1210T1310T1410T1510T1610T1710T1810T1910T20
11T111T211T311T411T511T611T711T811T911T10
11T1111T1211T1311T1411T1511T1611T1711T1811T1911T20
  • When picking colonies from the index plate, may have put two different colonies in 8T5 and 8T6.

PCR Cycles:

  • 95C @ 15 min
  • 39 cycles:
  1. 94C @ 30 sec
  2. 56C @ 30 sec
  3. 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)


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