Team:Cornell/Notebook/Salicylate reporter

From 2012.igem.org

(Difference between revisions)
(June 30th, Saturday)
(June 30th, Saturday)
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* Took PCR out of thermal cycler at 9:00 am (DPW)
* Took PCR out of thermal cycler at 9:00 am (DPW)
** Set up gel using NEB 100bp and 2-log ladders (10:00am)
** Set up gel using NEB 100bp and 2-log ladders (10:00am)
 +
<br>
* Continued gel extraction of p21 PCR digest from previous day (SS)
* 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)
* 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.
 +
<br>
 +
* 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.

Revision as of 22:51, 30 June 2012

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June

Week

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)


  • 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.