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From 2012.igem.org

June 29th, Friday

• Vent PCR at 11:00 (DPW)
  • Amplifying both previous Phusion PCR band and original p21 template
  • Dylan's magic triple anneal method (55/60/63)
• Gel purified PCR product from Phusion template (~1:20 pm) (DPW)
  • Quantified product at 22.4 ng/uL
  • Set up digestion of p21 PCR product with EcoRI-HF and AscI (~9:00 pm).
    • 22 ng/uL --> 45.5 uL sample for 1 ug digest
    • Buffer 4
  • Ran digestion on gel. (~11:00 pm)
  • Sliced out relevant band on gel, stored overnight at -20.

• Miniprepped overnight cultures of PL14-PL20 (~1:00 pm, STC)
  • Using C1015 rotor, 6666 x g, the Corning culture tubes only fit in the centrifuge with the lids off

• Made LB, 3x 60 mL in 100 mL bottles (~ 3:00pm, SS)
• Made LB Agar, 4 x 250 mL LB Agar in 500 mL bottles (~3:00, CS)
• Autoclaved LB, LB Agar, and milliQ (~3:30 pm, SS)
• Made LB plates with Kan (~6:30 pm, CMR)

• CUGEM movie outing at 8:00 pm.

• Phusion PCR at 10:00 pm (DPW)
  • Dylan's magic triple anneal method (57/65/70, 35 cycles total)
  • Amplifying nah operon from Gibson 1
  • Appending BioBrick cutsites for ligation into pSB3C5

June 30th, Saturday

• Took PCR out of thermal cycler at 9:00 am (DPW)
  • Set up gel using NEB 100bp and 2-log ladders (10:00am)
  • Gel extracted PCR product, quantified at ~10ng/uL
  • Set up new Phusion PCR using Gibson 1 as template
    • Dylan's magic three-anneal method (57.6/65/72)
    • Extension time of 3 min.

• Continued gel extraction of p21 PCR digest from previous day (SS)
• Set up ligation of p21 PCR digest extraction and dephosphorylated pBBRBB+mtrB (11:11am, SS)
  • Desalted ligation using Millipore membrane paper
  • Transformed 2 electrocompetent DH5alpha stocks at 5:30 pm and 5:50 pm, respectively.
  • Observed time constants for electroporation of 4.38 ms and 4.24 ms, respectively
  • Let cells recover for 1 hour, plated on LB + Kan.

• Set up two ligations of pSB3C5 into PNNL electrocompetent Shewanella strain JG700 (6:30 pm, Sp.C and St.C)
  • First transformation performed at usual PNNL voltage of 0.75 kV (time constant of 9.30 ms)
  • Second transformation performed at Myers and Myers specification of 0.55 kV (time constant of 9.34 ms).