Team:Cornell/Notebook/Salicylate reporter

From 2012.igem.org

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Write description
 
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==June==
 
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===Week===
 
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====June 29th, Friday====
 
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* [[Vent PCR]] at 11:00 (DPW)
 
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** Amplifying both previous Phusion PCR band and original p21 template
 
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** Dylan's magic triple anneal method (55/60/63)
 
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* Gel purified PCR product from Phusion template (~1:20 pm) (DPW)
 
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** Quantified product at  22.4 ng/uL
 
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** Set up digestion of p21 PCR product with EcoRI-HF and AscI (~9:00 pm).
 
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*** 22 ng/uL --> 45.5 uL sample for 1 ug digest
 
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*** Buffer 4
 
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** Ran digestion on gel. (~11:00 pm)
 
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** Sliced out relevant band on gel, stored overnight at -20.
 
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<br>
 
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* [[Miniprep]]ped overnight cultures of PL14-PL20 (~1:00 pm, STC)
 
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** Using C1015 rotor, 6666 x g, the Corning culture tubes only fit in the centrifuge with the lids off
 
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<br>
 
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* Made [[LB]], 3x 60 mL in 100 mL bottles (~ 3:00pm, SS)
 
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* Made [[LB|LB Agar]], 4 x 250 mL LB Agar in 500 mL bottles (~3:00, CS)
 
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* [[Autoclave]]d LB, LB Agar, and milliQ (~3:30 pm, SS)
 
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* Made [[LB]] plates with Kan (~6:30 pm, CMR)
 
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<br>
 
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* CUGEM movie outing at 8:00 pm.
 
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<br>
 
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* [[Phusion PCR]] at 10:00 pm (DPW)
 
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** Dylan's magic triple anneal method (57/65/70, 35 cycles total)
 
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** Amplifying nah operon from Gibson 1
 
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** Appending BioBrick cutsites for ligation into pSB3C5
 
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<br>
 
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====June 30th, Saturday====
 
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* Took PCR out of thermal cycler at 9:00 am (DPW)
 
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** Set up gel using NEB 100bp and 2-log ladders (10:00am)
 
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** Gel extracted PCR product, quantified at ~10ng/uL
 
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** Set up new [[Phusion PCR]] using Gibson 1 as template
 
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*** Dylan's magic three-anneal method (57.6/65/72)
 
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*** Extension time of 3 min.
 
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<br>
 
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* Continued gel extraction of p21 PCR digest from previous day (SS)
 
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* Set up ligation of p21 PCR digest extraction and dephosphorylated pBBRBB+mtrB (11:11am, SS)
 
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** Desalted ligation using Millipore membrane paper
 
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** Transformed 2 electrocompetent DH5alpha stocks at 5:30 pm and 5:50 pm, respectively.
 
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** Observed time constants for electroporation of 4.38 ms and 4.24 ms, respectively
 
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** Let cells recover for 1 hour, plated on LB + Kan.
 
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<br>
 
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* Set up two ligations of pSB3C5 into [[PNNL]] electrocompetent Shewanella strain JG700 (6:30 pm, Sp.C and St.C)
 
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** First transformation performed at usual PNNL voltage of 0.75 kV (time constant of 9.30 ms)
 
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** Second transformation performed at [[Myers and Myers]] specification of 0.55 kV (time constant of 9.34 ms).
 
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<br>
 
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====July 1st, Sunday====
 
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* Set up gel for electrophoresis (9:50am, DPW + CMR)
 
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** 1% gel in BIO-RAD Mini-Sub Cell system for continuation of ladder test using SYBR Green(10:50, CMR)
 
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*** Ran NEB 100 bp ladder, NEB 2-log ladder, Promega 1 kb ladder
 
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*** Ran at 100 V.
 
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** 1% gel in Owl box using ethidium bromide (10:50, DPW)
 
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*** Ran nah operon PCR product from previous night
 
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*** Ran at 55 V.
 
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<br>
 
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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.
 

Latest revision as of 16:24, 26 October 2012