Team:Duke/Notebook

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<a id="apr10"><h2> April 10 </h2></a>
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<div class="obj">Objective: Create Z4EV-dCas9-Mxi1 construct</div>
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<div class="ppl">Matthew Farnitano</div>
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Revision as of 17:31, 28 July 2014


April 7

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw

PCR of Z4EV from pMN10

  • new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM)
  • 4 tubes with 0, 0.3, 0.6, 1 uL template
    • iGEM 3-step protocol with 65C anneal, 20 sec extend
  • Note: did not dilute template beforehand (do this in future)

Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)

  • Expected band size 715 bp
  • Control (no template) showed no band, lane 4 produced faint band
  • Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)

Next steps:

  • Run on gel to confirm (4/8)
  • digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8)

April 8

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

Agarose Gel with Z4EV PCR products

  • PCR from 04/07/2014 of Z4EV from pMN10
  • Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)
    • Expected band size 715 bp
    • Control (no template) showed no band, lane 4 produced faint band
    • Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)

PCR cleanup of Z4EV PCR products

  • PCR from 04/07/2014 of Z4EV from pMN10
  • Used only lanes 2 and 3 (successful from gel)
  • Concentration 32.4 ng/uL in 50 uL

Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1

  • Both with SpeI-HF and NcoI-HF in Cutsmart
  • PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction
  • dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction

PCR cleanup of Z4EV PCR digest product

  • 04/08 digest of 04/07 PCR
  • Results: Final concentration negligible, no DNA

Next steps:

  • Z4EV PCR again from pMN10
  • Digest of PCR in SpeI/NcoI
  • Gel extraction of digested TDH3-dCas9-MxiI

April 10

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

Curabitur a dui cursus libero cursus sodales nec lobortis leo. Nulla tempor volutpat eros, id tristique felis pellentesque nec. Etiam ultrices ligula ut eros viverra, eu pharetra lacus volutpat. Aliquam mattis lobortis leo, sed ornare lacus. Mauris vel gravida velit. Quisque ut viverra nisl, eget consectetur enim. In commodo imperdiet euismod. In rhoncus a enim ut sollicitudin. Praesent mattis dolor a erat euismod interdum. In a neque non enim sollicitudin fringilla quis sit amet justo. Nam mauris massa, tristique sit amet semper sit amet, pharetra eget felis. Sed in tortor purus. Donec elit nunc, commodo vel mollis in, iaculis id nunc. Praesent ut convallis dui, a bibendum dui. Donec non malesuada quam. Duis sem urna, tincidunt sit amet odio sed, gravida tincidunt enim. Cras risus elit, laoreet vitae feugiat non, viverra vel eros. Suspendisse potenti. Maecenas vel suscipit eros.

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