Team:Cornell/Dylan Scratch Notebook3

From 2012.igem.org

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<p class="heading"> August 21st, Tuesday </p>
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Summary goes here.
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<p class="heading1"> Click to view the details. </p>
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<li type=square><a href="https://2012.igem.org/Team:Cornell/Protocols/Miniprep"> Miniprepped </a> directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
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<li type=square>Ran undigested miniprep with <a href="https://2012.igem.org/Team:Cornell/Protocols/Gel_electrophoresis"> gel electrophoresis</a>, 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 <a href="https://2012.igem.org/Team:Cornell/Protocols/Sequencing"> sequencing</a>.</ul>
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<img src="https://static.igem.org/mediawiki/2012/a/a1/Cornell2012_0621_Gibson-entire-plasmid.jpg" alt="Supercoiled Gibson" width="600" />
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<p class="heading"> August 21st, Tuesday </p>
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Reactor stuff.
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More thorough.
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<p class="heading1"> Click to view the details. </p>
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Dylan connected second reactor to SP-200, which just arrived back from field use in Alaska. Current was steady at ≈ 20 µA. However, flow through the reactor was paused because the media tank we depleted. Dylan made new media, and will wait overnight to ensure that the reactor reaches steady state. If steady state is indeed reached, we will perturb the reactors with salicylate to start getting more definitive data.
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Latest revision as of 00:20, 22 August 2012




Team:Cornell - Experimental Style Template


Overview

During the summer, we began work by holding a Synthetic Biology Bootcamp at the Delisa Lab.

June

June 12th-16th

Peace, Love, and Pipettes.

June 12th, Tuesday

Today, we began bootcamp!
A more thorough summary goes here!

Click to view the details.

These are more details. These are more details. These are more details. These are more details. These are more details. These are more details. These are more details. These are more details. These are more details. These are more details.


June 13th, Wednesday

Summary goes here.

Click to view the details.

  • Set up 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.
  • 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.
  • Work done by: Caleb, Claire, Dylan, Spencer, Steven, and Swati


June 14th, Thursday

Summary goes here.

Click to view the details.

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 PCR with shorter extension time.


June 15th, Friday

Summary goes here.

Click to view the details.

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.


iGEM.

June 16th, Saturday

Summary goes here.

Click to view the details.



June 17th-23rd

June 17th, Sunday

Summary goes here.

Click to view the details.

Successful PCR of pBMT-1 gel purified .


June 18th, Monday

Summary goes here.

Click to view the details.



June 19th, Tuesday

Summary goes here.

Click to view the details.

  • Ran Gibson Assembly of nah operon fragments into pBMT-1 backbone and transformed into DH5α electrocompetent E. coli cells.
  • Set up PCR to amplify the Gibson Assembly products.
  • Work done by: Dylan and Swati


June 20th, Wednesday

Summary goes here.

Click to view the details.

  • Set up a digestion of the PCR of Gibson Assembly products and ran the digested products on a gel to see if Gibson worked.
  • Three colonies on plates of DH5α transformed with Gibson Assembly product. Made liquid cultures to miniprep and sequence.
  • Work done by: Dylan and Swati


June 21st, Thursday

Summary goes here.

Click to view the details.



June 22nd, Friday

Summary goes here.

Click to view the details.

  • Miniprepped directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
  • Ran undigested miniprep with 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 sequencing.
Supercoiled Gibson


August 21st, Tuesday

Summary goes here.

Click to view the details.

  • Miniprepped directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
  • Ran undigested miniprep with 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 sequencing.
Supercoiled Gibson


August 21st, Tuesday

Reactor stuff.
More thorough.

Click to view the details.

Dylan connected second reactor to SP-200, which just arrived back from field use in Alaska. Current was steady at ≈ 20 µA. However, flow through the reactor was paused because the media tank we depleted. Dylan made new media, and will wait overnight to ensure that the reactor reaches steady state. If steady state is indeed reached, we will perturb the reactors with salicylate to start getting more definitive data.





June 22nd, Friday

  • Miniprepped directly transformed Gibson Assembly product for sequencing using the the Gibson nah1F and nah4R primers (each w/ 20 bp overhangs).
  • Ran undigested miniprep with 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 sequencing
Cornell2012 0621 Gibson-entire-plasmid.jpg

June 23rd, Saturday

June 24th-30th

June 24th, Sunday

June 25th, Monday

June 26th, Tuesday

June 27th, Wednesday

Rafael and Dylan making an agarose gel.

June 28th, Thursday

Stephen and Archie Gel purifying our arsR construct
  • Gel purified arsR construct

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).


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