Team:Duke/Notebook/April

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Revision as of 16:02, 29 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

April 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

April 15

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.

April 16

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Charlie Cooper

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