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

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dspB Track

  • Transformants taken out at 0940
  • Plate results:
Transformant Results
Plate # of colonies
1no colonies
37 colonies
419 colony
pBADTMTC colonies

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 Master mix
Reagent1x rxn volume (uL)Master Mix
5x rxn buffer5x1155
10mM dNTP2x1122
sdH2O10.55x11116.05
Phusion polymerase0.2x112.2
MgCl22x1122
DMSO - 5%1.25x1113.75
10uM fw primer (G1004)2x1122
10uM re primer (G1005)2x1122
Total25275
PCR Tubes
From plate pBAD:pT1pT2
From plate 3:3T13T23T3
From plate 4:4T14T24T34T4
W (H2O control)

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

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

Gel orientation:

Gel orientation
pT1pT23T13T23T3100bp ladder4T14T24T34T4W (control)

Results:

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

  • Picked colonies 3T1 & 4T3 into 2 tubes
  • Put in 37C incubator @ 1606

Vicki Ma 13:56, 11 August 2010 (EDT)

Phage/Phage Standard Track

Eric F.

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 Track

Eric F. + Melody Wang

Biofilm Growth Protocol

  • 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

Plan:

  • 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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