Team:Duke/Notebook

From 2014.igem.org

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<p> PCR of Z4EV from pMN10</p>
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<li>Repeat of PCR from 4/7/14</li>
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<li>Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p</li>
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<li>Diluted pMN10 template to 1 ng/uL before use</li>
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<li>8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction</li>
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<ul><li>iGEM 3-step protocol with 65C anneal, 20 sec extend</li></ul>
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<li>Results (see 4/11): expected bands present, but concentrations too low</li>
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<p> Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest</p>
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<li>Digest performed 4/8 with SpeI/NcoI</li>
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<li>Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)</li>
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<li>Obtained consistent band sizes, used 400 mg gel in extraction <</li>
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<li>Gel picture: <img src="https://static.igem.org/mediawiki/2014/3/3c/4-10-14gel.png"></li>
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<li> Results: obtained 20 uL product at 55 ng/uL (froze for later use)</li>
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<p> Next steps: </p>
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<li> Run gel of PCR</li>
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<li> Clean up PCR</li>
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Revision as of 17:37, 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

PCR of Z4EV from pMN10

  • Repeat of PCR from 4/7/14
  • Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p
  • Diluted pMN10 template to 1 ng/uL before use
  • 8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction
    • iGEM 3-step protocol with 65C anneal, 20 sec extend
  • Results (see 4/11): expected bands present, but concentrations too low

Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest

  • Digest performed 4/8 with SpeI/NcoI
  • Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)
  • Obtained consistent band sizes, used 400 mg gel in extraction <
  • Gel picture:
  • Results: obtained 20 uL product at 55 ng/uL (froze for later use)

Next steps:

  • Run gel of PCR
  • Clean up PCR
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