Week 8:
07/25/2011
1. anneal tetR and GFP
2. digest tetR, GFP and CRISPR
3. PCR amplification of CAS3
4. Use PCDF2, PCOLA to digest, ligate with CASO by EcoRI and NotI
07/26/2011
1. Grow 12 colonies from CM+tetR plate
2. Scan transformed sample from yesterday, grow 24colonies (12 from CRISPR-GFP and 12 from CRISPR-tetR), inoculation then
3. Inoculate 12 colonies from PCOLA with CASO plate
4. Transform PCDF with CASO into DH5α cells
5. Gel verify CASO
6. Plated PCDF with CASO
7. Run a gel for CAS3 and CASO plasmids
8. Reamplify CAS3 with taq
07/27/2011
1. Mini-prep all 12 colonies from CM+tetR plate
2. Digest with EcoRI and PstI
3. Transform into tetO::GFP competent cells
4. Run a gel for PCR taq product CAS3
5. Purify the CAS3
6. Mini-Prep12 from CRISPR-GFP and 12 from CRISPR-tetR, and PCOLA with CASO
07/28/2011
1. Inoculate tetR-GFP/BL21
2. Digest PCDF+PCOLA
3. Blunt end CAS3
4. Check if the tetR and GFP spacers were successfully inserted into CRISPR by digesting with enzyme and running a gel, we are
cutting with NocI and BamHI, if the plasmid does not have the spacer, it will not cut at all
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