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Team:Cornell/Notebook/Salicylate reporter
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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)
- 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 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.
- 1% gel in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green(10:50, CMR)
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.
