IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/13

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dspB Track
PCR off gDNA for more PCR products


 * To A1,A2,A3: add 2uL of up-his & dw primers each
 * To B1,B2,B3: add 2uL of up & dw primers each

PCR Cycles:
 * 98C @ 3min
 * Cycle 27x:
 * 98C @ 10 sec
 * 72C @ 30 sec
 * 72C @ 40 sec
 * 72C @ 10 min
 * 10C @ hold

Start: 0907 End: 1010

Gel verification colony PCR products
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Changes: 1% agarose gel
 * Machine conditions: 0.5x TBE buffer, 100V, 45min

O/N cultures

 * 2 tubes (3T1, 4T3) taken out at 0950

Miniprep

 * Miniprepped 3T1, 4T3
 * Protocol: in SOP binder (Alkaline lysis)

Restriction Digest on miniprepped DNA

 * See RD protocol (BioBricks)

Gel Verification on mini-prepped RD
Gel orientation: Results:
 * Protocol: gel verification protocol in Protocol (SOP)
 * Changes: 1% agarose gel
 * Machine conditions: 0.5x TBE buffer,

Plan: RD + ligation + transformation

Restriction Digest
Protocol: Biobrick Protocol


 * DNA: add 5uL of backbone + 2uL of insert each
 * Enzymes: EcoRI and SpeI (1uL each to each tube)


 * DNA: add 5uL of backbone + 2uL of insert each
 * Enzymes: EcoRI and SpeI (1uL each to each tube)


 * RD Tubes:
 * AA, TA, CA, AB, TB, CB, 1-16 all contain insert dspB His
 * AC, TC, CC< AD, TD, CD, 17-32 all contain insert dspB no-His

Ligation
Calculations $$ ratio \times \frac{insert \rm length}{vector \rm length} \times vector \rm mass = insert \rm mass (ng)$$ $$3 \times \frac{1200}{2400} \times =  ng$$ $$1uL \rm vector\times\frac{ng \rm vector}{1uL vector}\times\frac{ng \rm insert}{ng \rm vector}\times\frac{uL \rm insert}{x ng \rm insert} = $$ Vicki: ligation - 6:1 ratio using
 * Where x = concentration of insert
 * 1) amp backbone
 * 2) amp+tet backbone
 * 3) chlor backbone

Marianne: Ligation - chlor backbone using ratios:
 * 2:1,3:1,4:1,6:1,7:1,8:1,9:1,10:1

Transformation

 * Protocol: Transformation protocol from SOP
 * Changes: 10uL of ligation mix instead of 1uL; 1 hour incubation instead of 2 hours

8325-4 Biofilm Growth
Streaked a TSA plate with 8325-4 TSB O/N culture and put in incubator at 37°C

RN4220 Biofilm Growth
Completed Day 3 of the protocol for both plates
 * Test #1 - control E5 showed growth
 * Test #2 - controls B2, B11, C5, E7 and G2 showed growth
 * We decided to continue the experiment as a wet run (due to the contamination the results are meaningless)
 * Next time we will perform the inoculation step in the bio safety cabinet
 * The 60°C air dry step was completed using a heat block
 * For the overnight step, we placed the plates in the bio safety cabinet


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