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| - | {{:Team:Cornell/Navbar}}
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| - | {{:Team:Cornell/TOC|right}}
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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.
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