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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
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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