Team:Duke/Notebook

From 2014.igem.org

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</div>
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<div class="day">
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<a id="apr13"><h2>April 13 - Unfinished</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 and Garima Tomar</div>
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<div class="lab">
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<p> PCR of Z4EV from pMN10</p>
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<ul>
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<li>Third attempt (previously 4/7, 4/10)</li>
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<li>oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p</li>
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<li>8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction</li>
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<ul>
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<li>Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction</li>
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<li>iGEM 3-step protocol with 65C anneal, 1 min extension</li>
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</ul>
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<li>Changes from previous: more template, more polymerase, longer extension</li>
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</ul>
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<p> Results (Date): </p>
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<ul>
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<li>???</li>
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</ul>
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<p> Next steps: </p>
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<ul>
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<li>Run on gel</li>
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<li>Clean up PCR with new notes (see 4/11), see if concentration is better </li>
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</ul>
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</div>
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</div>
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04/14/14
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Objective: Create Z4EV-dCas9-Mxi1 construct
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Matthew Farnitano
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Agarose gel of Z4EV PCR
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Z4EV from pMN10, 4/13 PCR
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2-log ladder and 8 identical reaction tubes
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Expected band size ~715 bp
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All lanes showed band 700-800 bp
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band intensity much greater than previous attempts
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Unable to take normal picture: used dark room and iPhone camera to photograph
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PCR cleanup of Z4EV PCR from 4/13
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Qiagen miniprep steps 7-10
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8 tubes into 1 final product
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Modifications to protocol:
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ran PB buffer through twice
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let column sit, covered, for 10-15 min after PE buffer wash/discard step
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let water for elution sit on column for 3 min before spin
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Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use)
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Next Steps:
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Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI
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ligation of Z4EV with dCas9 backbone (obtained 4/10)
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transformation
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04/15/14
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 +
Objective: Create Z4EV-dCas9-Mxi1 construct
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Matthew Farnitano
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Restriction Digest of Z4EV-PCR with SpeI/NcoI
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PCR from 4/13, cleaned 4/14
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Cut with SpeI-HF and NcoI-HF in Cutsmart buffer
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30 uL PCR product (407.5 ng/uL) in 50 uL reaction
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Incubated at 37C for 4.5 hrs
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PCR Cleanup of Restriction Digest of Z4EV
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Qiagen kit with modified methods described on 4/14
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Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop)
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Ligation of Z4EV PCR and dCas9-MxiI backbone
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Z4EV from pMN10, digested with NcoI and SpeI (4/15)
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dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10
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3:1 molar ratio backbone:insert
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20 uL total reaction
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15.3 uL backbone (55 ng/uL)
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0.7 uL insert (248.1 ng/uL)
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Left in cold room overnight in 16C heat block
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Next steps:
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Transform ligated plasmid into E. coli, clone, then miniprep.
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 +
04/16/14
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Objective: Create Z4EV-dCas9-Mxi1 construct
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Matthew Farnitano
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Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli
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Ligation left overnight for 22 hrs
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Heat shock protocol with chemically competent E. coli
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1 sample plus 1 no-insert control
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grown in SOC medium, plated on LB+Amp plates
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Results (Charlie Cooper) (4/17): No colonies on either plate
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Possibly used ineffective cells
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Next steps:
 +
Digest and gel extraction of backbone
 +
Ligation and Transformation
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Revision as of 17:58, 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

April 11
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw

Agarose gel to analyze PCR of Z4EV from pMN10

  • PCR from 4/10
  • Expected band size: 715 bp
  • Showed band at expected size in all seven non-control lanes
  • Intensities appear low, increase in higher lanes (higher template conc.)
  • Gel picture:

PCR cleanup of Z4EV from 4/10 PCR

  • final concentration from 7 tubes: 25.8 ng/uL in 30 uL
  • Need to improve

Next Steps:

  • Redo PCR of Z4EV. Things to try:
    • Use previous cleanup as a template
    • use 1 uL enzyme instead of 0.5
    • slightly longer extension time
    • higher template concentration
  • PCR cleanup: Things to try:
    • Run PB buffer flow-through back through filter during step 7
    • let PE buffer sit in column (covered) while ethanol evaporates
    • let elution buffer sit on DNA for 2-3 minutes before spin

April 13 - Unfinished
Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Garima Tomar

PCR of Z4EV from pMN10

  • Third attempt (previously 4/7, 4/10)
  • oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p
  • 8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction
    • Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction
    • iGEM 3-step protocol with 65C anneal, 1 min extension
  • Changes from previous: more template, more polymerase, longer extension

Results (Date):

  • ???

Next steps:

  • Run on gel
  • Clean up PCR with new notes (see 4/11), see if concentration is better
04/14/14 Objective: Create Z4EV-dCas9-Mxi1 construct Matthew Farnitano Agarose gel of Z4EV PCR Z4EV from pMN10, 4/13 PCR 2-log ladder and 8 identical reaction tubes Expected band size ~715 bp All lanes showed band 700-800 bp band intensity much greater than previous attempts Unable to take normal picture: used dark room and iPhone camera to photograph PCR cleanup of Z4EV PCR from 4/13 Qiagen miniprep steps 7-10 8 tubes into 1 final product Modifications to protocol: ran PB buffer through twice let column sit, covered, for 10-15 min after PE buffer wash/discard step let water for elution sit on column for 3 min before spin Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use) Next Steps: Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI ligation of Z4EV with dCas9 backbone (obtained 4/10) transformation 04/15/14 Objective: Create Z4EV-dCas9-Mxi1 construct Matthew Farnitano Restriction Digest of Z4EV-PCR with SpeI/NcoI PCR from 4/13, cleaned 4/14 Cut with SpeI-HF and NcoI-HF in Cutsmart buffer 30 uL PCR product (407.5 ng/uL) in 50 uL reaction Incubated at 37C for 4.5 hrs PCR Cleanup of Restriction Digest of Z4EV Qiagen kit with modified methods described on 4/14 Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop) Ligation of Z4EV PCR and dCas9-MxiI backbone Z4EV from pMN10, digested with NcoI and SpeI (4/15) dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10 3:1 molar ratio backbone:insert 20 uL total reaction 15.3 uL backbone (55 ng/uL) 0.7 uL insert (248.1 ng/uL) Left in cold room overnight in 16C heat block Next steps: Transform ligated plasmid into E. coli, clone, then miniprep. 04/16/14 Objective: Create Z4EV-dCas9-Mxi1 construct Matthew Farnitano Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli Ligation left overnight for 22 hrs Heat shock protocol with chemically competent E. coli 1 sample plus 1 no-insert control grown in SOC medium, plated on LB+Amp plates Results (Charlie Cooper) (4/17): No colonies on either plate Possibly used ineffective cells Next steps: Digest and gel extraction of backbone Ligation and Transformation

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