Team:Cornell/Notebook/Salicylate reporter

From 2012.igem.org

Revision as of 15:04, 2 July 2012

Write description

June

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).

July

July 1st, Sunday

• Set up gel for electrophoresis (9:50am, DPW + CMR)
  • 1% gel in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green(10:50, CMR)
    • Ran NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder
    • Ran at 100 V.
  • 1% gel in Owl box using ethidium bromide (10:50, DPW)
    • Ran nah operon PCR product from previous night
    • Ran at 55 V.

Because we learned that our SYBR Green was causing ladder to run strangely, Dylan decided to redo a Vent PCR to amplify the salicylate reporter region out of p21.
Liquid culture of JG700.
Replated p21, p22, JG700, JG1220, JG1537, JG1219

July 2nd, Monday

Today, Dylan and Caleb ran a gel of a Vent PCR of p21 (the PCR product being the salicylate reporter), then gel extracted the appropriately-sized fragment, and finally digested the gel extracted salicylate reporter with EcoRI and AscI.