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| - | {{:Team:Cornell/Navbar}}
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| - | {{:Team:Cornell/TOC|right}}
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| - | =June=
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| - | ==June 10th-16th==
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| - | ===June 13th, Wednesday===
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| - | *Set up [[Team:Cornell/Notebook/Phusion_PCR|PCR]] for four fragments of the nah operon as well as the pBMT-1 backbone, using primers designed to mutate cut-sites concurrently with Gibson Assembly. Also set up a PCR for the entire nah operon. Due to length of the fragments, a longer extension time was chosen.
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| - | *If Gibson Assembly fails, but we are able to PCR the entire 10kb nah operon, an alternate method of mutation using three sequential site-directed mutageneses will be pursued.
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| - | **''Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati''
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| - |
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| - | ===June 14th, Thursday===
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| - | *PCR only amplified nah1, nah3, and nah4 - longer fragments (nah2, the full nah operon, and pBMT-1) were not identified on the PCR product gel. Set up another [[Team:Cornell/Notebook/Phusion_PCR|PCR]] with shorter extension time.
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| - |
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| - | ===June 15th, Friday===
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| - | *PCR of four out of five products visible on gel. Set up PCR of pBMT-1, final fragment required for Gibson Assembly of the nah operon.
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| - | ==June 17th-23rd==
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| - | ===June 17th, Sunday===
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| - | *Successful PCR of pBMT-1 [[Team:Cornell/Notebook/Gel_purification| gel purified]].
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| - | ===June 19th, Tuesday===
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| - | *Ran [[Team:Cornell/Notebook/Gibson_assembly|Gibson Assembly]] of nah operon fragments into pBMT-1 backbone and [[Team:Cornell/Notebook/Transformation_ecoli | transformed]] into DH5a electrocompotent ''E. coli'' cells.
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| - | *Set up [[Team:Cornell/Notebook/Phusion_PCR|PCR]] to amplify the Gibson Assembly products.
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| - | **''Work done by: Dylan and Swati''
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| - | ===June 20th, Wednesday===
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| - | *Set up a [[Team:Cornell/Notebook/Digestion|digestion]] of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
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| - | *Three colonies on plates of DH5a transformed with Gibson Assembly product. Made liquid cultures to miniprep and sequence.
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| - | **''Work done by: Dylan and Swati''
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| - |
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| - | ===June 22nd, Friday===
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| - | *[[Team:Cornell/Notebook/Miniprep | Miniprepped]] directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
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| - | *Ran undigested miniprep with [[Team:Cornell/Notebook/Gel_electrophoresis|gel electrophoresis]], looking for large bands corresponding to supercoiled DNA. The gel shows distinct bands for all three lanes. We interpreted this to mean that we got product. Submitted for [[Team:Cornell/Notebook/Sequencing|sequencing]]
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| - | ::[[File:Cornell2012_0621_Gibson-entire-plasmid.jpg|600px]]
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| - | ==June 24th-30th==
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| - | ===June 27th, Wednesday===
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| - | [[Image:2012_627_RafDylanGelMaking.jpg|thumb|right|Rafael and Dylan making an agarose gel.]]
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| - | *Ran digest of gibson-assembled nah operon with [[Team:Cornell/Notebook/Gel_electrophoresis|gel electrophoresis]]
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| - |
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| - | ===June 28th, Thursday===
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| - | [[Image:2012_6_28_StevenArchieGelLoading.jpg|thumb|right|Stephen and Archie Gel purifying our arsR construct]]
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| - | *Gel purified arsR construct
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| - |
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| - | ===June 29th, Friday===
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| - | * [[Team:Cornell/Notebook/Vent_PCR|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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| - | * [[Team:Cornell/Notebook/Miniprep|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 [[Team:Cornell/Notebook/LB|LB]], 3x 60 mL in 100 mL bottles (~ 3:00pm, SS)
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| - | * Made [[Team:Cornell/Notebook/LB|LB Agar]], 4 x 250 mL LB Agar in 500 mL bottles (~3:00, CS)
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| - | * [[Team:Cornell/Notebook/Autoclave|Autoclave]]d LB, LB Agar, and milliQ (~3:30 pm, SS)
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| - | * Made [[Team:Cornell/Notebook/LB|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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| - | * [[Team:Cornell/Notebook/Phusion_PCR|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 [[Team:Cornell/Notebook/Phusion_PCR|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 [[Team:Cornell/Notebook/Transformation_shewanella|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 [[Team:Cornell/Notebook/Transformation_shewanella|Myers and Myers]] specification of 0.55 kV (time constant of 9.34 ms).
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| - | <br>
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| - | =July=
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| - |
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| - | =August=
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