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)
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
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