Week 6:
07/11/2011
1. inoculate prs 423+MET25, prs 424+MET25, prs 424+MET25
2. PCR TPS1, MSN2, ELO1, OLE1
3. Inoculate and mini-prep:
PCDF(s) and PCOLA(K)
07/12/2011
1. Observations: PCR products of CASO didn’t work
2. Test:
Old forward primer with new reverse primer
New forward primer with old reverse primer
3. Purify ligated vectors:
prs 423+MET25, prs 424+MET25, prs 424+MET25
4. Run a gel for PCR genes
5. Digest ligated vectors and run a gel to test them
Observations: ligated products didn’t work, all genes work
07/13/2011
1. Make competent cells BL21
2. Transformation and inoculation :
3. 1124, 1126, 1125, 1128, 1129, 1130, PCDF(5 µL step) and PCDF(10 µL step)
4. Run a gel for CAS3 and PCDF
5. Purify DNA:
PCDF, CRISPR, pSB1C3, tet spacer, GFP spacer, CAS3 #1 and CAS3 #2
6. PCR CASO with E, E1, E2 and AP1 and AP2 (primers)
07/14/2011
1. Make competent cells , check the OD after incubating in shaker until OD~0.25
Check every 30 mins
2. Inoculate and transform PCOLA
3. Transform CRISPR
4. Mini-prep 1124-1130, PCDF
5. Gel verify CASO by PCR
6. Inoculate DH5α and BL21
7. Digest tetR
07/15/2011
1. Transform pSB1C3 tetR, CRISPR tetR, CRISPR GFP, CRISPR
2. Run a gel to test:
Ladder, CAS3 #2, CAS3 #3, PCDF, CRISPR, tetR spacer, GFP spacer, pSB1C3
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