This is what we were trying to accomplish in April.

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)

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

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

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

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

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

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.

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

This is what we were trying to accomplish in May and June.

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

• Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

• Transformation

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

• Followed Charlie’s Cloning Protocols
• Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
• Plated the transformed DNA and put in incubator at 37C
• Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

• Look at plates, compare cultures