IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/08/10

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dspB Track

 * Transformants taken out at 0940
 * Plate results:

Colony PCR
Protocol: See common protocol
 * Changes: use Phusion pol; add MgCl2 and DMSO
 * Samples to be PCRed:
 * 2 from pBAD
 * 3 from plate 3
 * 4 from plate 4
 * 1 H2O control
 * Total: 10 samples + 1 extra = 11 samples

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

Start: 1201 End: 1305

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, 60min

Transformation

 * Transform 1 and 2 (ligation mixes) from yesterday since there were no colonies on 1 (yesterday) and 2 (4 days ago) --> currently no transformants with His-tag primers
 * Protocol: transformation protocol from SOP
 * Changes: 10uL of ligation mix instead of 1uL; in 37C incubator for 1.5 hours
 * Plated on amp plates and into incubator (37C) @ 1445

ON Culture for miniprep
Vicki Ma 13:56, 11 August 2010 (EDT)
 * Picked colonies 3T1 & 4T3 into 2 tubes
 * Put in 37C incubator @ 1606

Prep

 * Both O/N culture controls showed no growth
 * All bacterial samples showed growth
 * Melted Soft+Hard Agar made yesterday
 * Soft: 10x40mL Falcon tube aliquots
 * Hard: 20x20mL Plates

UV Induction Protocol

 * Used SOP finalized yesterday (August 9th) - Strain = 8325-4
 * Growth of 1/50 dilution began @ 1108
 * No growth control was used in this step due to a lack of flasks aka bad planning
 * The 1/50 dilution was 31mL LB Broth w/ 620 microlitres of overnight culture
 * Wanted a t=0 reading and 30mL total volume during growth
 * t=0 OD600=0.154 using LB Broth as a blank
 * Note: Reading may have be done wrong
 * Inferring from RN4220 growth curve, OD600=0.6 should be at 1243
 * Planned to take reading at 1230
 * Took reading at 1240 - OD600 = 1.543
 * Began another dilution - hindered by lack of flasks
 * Discovered the strain we were working with is 8325-4 NOT 8325
 * 8325 has 3 prophages
 * 8325-4 has been cured of all 3 prophages
 * Experiment was scrapped due to strain mixup

Biofilm Growth Protocol
Plan:
 * Refined the biofilm protocol to a work of art
 * Each person (Melody + Eric) began 2 O/N cultures and 1 control
 * Overnight cultures were in 3mL of TSB broth in 10mL test tubes
 * Strain 1: 8325-4
 * Strain 2: RN4220
 * RN4220 was picked off a TSA plate and will therefore be used to make a biofilm on August 11th
 * 8325-4 needs to be spread on a TSA plate before the biofilm growth protocol can start
 * Will spread the O/N culture onto a TSA Plate
 * All O/N cultures in @ 1745 @ 220 RPM @ 37°C Eric Finlay 12:42, 11 August 2010 (EDT)


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