Week 10:
08/15/2011
1. Check the transformation of CRISPR+tetR and CRISPR+GFP
2. Inoculate 12 colonies of each, leave them in the shaker
3. Digest CAS3/PCOLA #12 and CASO/PCDF #4, incubate in 37c overnight
08/16/2011
1. Mini-prep all 24 inoculated colonies
2. PCR 12 plasmids of each (GFP+tetR)
Use mixture as follows:
A: GFP spacer Forward+T7 terminator
B: tetR spacer Forward+T7 terminator
C: GFP spacer Reverse+T7 promoter
D: tetR spacer Reverse+T7 promoter
3. Dephosphorylate CAS3/PCOLA
4. Column purify CAS3/PCOLA
5. Run a gel to test CASO/PCOLA
Observation: it didn’t work
08/17/2011
1. Remove overnight PCR and run a gel to check contents of PCR
2. Diluted PCDF+CASO #4 in tetO/R competent cells into 3 different tubes
Notes: each tube had a different combination of contributions to check if PCDF+CASO, tetO/R or both survived
3. Run a gel to test the 24 PCR samples
Observations: from the gel picture, we will pick #2,4,5,6 in GFP::CRISPR, and #1,3,4,5 in tetR::CRISPR, they look clear and reasonable
4. Inoculate the picked samples
08/18/2011
1. Take tube #2, 4, 5, 6
Notes: 6 tubes of LB for each, add 5mL LB, 250 µL of culture, add 5 µL antibiotics, add IPTG 2 hours later after inoculation. Make the
reaction mixture as follows:
| A | B | C | D | E | F |
| Amp | + | + | | | + | + |
| CM | | | + | + | + | + |
| IPTG | | + | | + | | + |
2. Freeze down the leftover cultures in 4 tubes
08/19/2011
1. Take CRISPR+GFP with tetO::GFP competent cells, redilute, adding Am and CM, reaction mixture is as follows:
5 µL culture+5mL LB+5 µL Am+5 µL CM
5 µL culture+5mL LB+5 µL Am+5 µL CM+5 µL IPTG
Notes: add IPTG right away
2. Mini-prep CASO+PCDF plasmid only with strep
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