Week 13:
09/06/2011
1. Digest/ ligate casO+PCDF
2. Transform ligated products into BL21 with tetO::GFP
3. Transform CRISPR GFP#7 into BL21 with tetO::GFP
4. Inoculate HT5 with/without IPTG
5. Inoculate HT115+tetO::GFP
6. Purify casO (PCR products)
7. Measure the DNA of casO::PCDF
09/07/2011
1. Make competent cells as follows:
BL21 (tetO::GFP, CRISPR::GFP)
HT115(tetO::GFP, CRISPR::GFP)
HT115(tetO::GFP)
2. Take OD reading of HT115 with/ without IPTG
3. Digest/ligate casO+PCDF
4. Transform CRISPR#7 into tetO cells(BL21)
5. Take equal amount of E.Coli, miniprep, transform into new DH5α, select on amp.
09/19/2011
1. Growth test, grow all the bacteria overnight
2. PCR cas3+casO from gDNA
09/08/2011
1. OD reading
2. Digest/ligate reaction
tetR with pSB1k3
tetO with pSB1k3
casO with PCDF
Transform into DH5α, and also IPTG samples into DH5α
09/09/2011
1. Make BL21(tetO, CRISPR-GFP #7) competent and make freeze stock
2. Make HT115(tetO, CRISPR-GFP #7) competent and make freeze stock
3. Make HT115(CRISPR-GFP #7) competent and make freeze stock
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Week 14:
09/13/2011
1. Check quality of gDNA
2. Make HT115 + BL21 (tetO, CRISPR-GFP #7) competent cells, freeze stock
3. Miniprep PCDF+casO
C. Plate 4 Well 16O (LovTAP Composite)
4. Digest tetO/tetR
09/15/2011
1. Run a gel of all minipreped PCDF+casO
2. PCR HT115, DH5α2, DH5α3 with primers
09/18/2011
1. Inoculate the following:
BL21 tetO::GFP
BL21 tetO::GFP, CRISPR::GFP#7
BL21 tetO::GFP, CRISPR::GFP#7, cas3
HT115 tetO::GFP
HT115 tetO::GFP, CRISPR::GFP#7
HT115 tetO::GFP, CRISPR::GFP#7, cas3
HT115 CRISPR::GFP #7
09/19/2011
1. Growth test, grow all the bacteria overnight
2. PCR cas3+casO from gDNA
09/20/2011
1. Make biobrick of cas3 and casO
2. Run a gel for cas3 and casO
3. Purify cas3 and casO
4. New experiment: dilution the bacteria to 1000 and 5000 cells and plate them
09/21/2011
1. Do growth test for the following:
BL21 tetO::GFP-IPTG
BL21 tetO::GFP+IPTG
BL21 tetO::GFP, CRISPR::GFP#7-IPTG
BL21 tetO::GFP, CRISPR::GFP#7+IPTG
BL21 tetO::GFP, CRISPR::GFP#7, cas3-IPTG
BL21 tetO::GFP, CRISPR::GFP#7, cas3+IPTG
BL21 tetO::GFP, CRISPR::GFP#7, cas3+IPTG an hour later
HT115 tetO::GFP-IPTG
HT115 tetO::GFP+IPTG
HT115 tetO::GFP+IPTG an hour later
HT115 tetO::GFP, CRISPR::GFP#7-IPTG
HT115 tetO::GFP, CRISPR::GFP#7+IPTG
HT115 tetO::GFP, CRISPR::GFP#7+IPTG an hour later
HT115 tetO::GFP, CRISPR::GFP#7, cas3-IPTG
HT115 tetO::GFP, CRISPR::GFP#7, cas3+IPTG
HT115 tetO::GFP, CRISPR::GFP#7, cas3+IPTG an hour later
HT115 CRISPR::GFP #7-IPTG
HT115 CRISPR::GFP #7+IPTG
HT115 CRISPR::GFP #7+IPTG an hour later
BL21 tetO::GFP, CRISPR::GFP#7, cas3
HT115 tetO::GFP
HT115 tetO::GFP, CRISPR::GFP#7
HT115 tetO::GFP, CRISPR::GFP#7, cas3
HT115 CRISPR::GFP #7
2. Dilution: plate on Amp #4 and #5 +/- IPTG, #4 and #5 have different OD values. Dilute 1000* and 5000* to both of them
3. Digest pSB1C3 and tetO/R vector
4. Transform casO#1, #2, #1, #2 on CM plates
09/22/2011
1. Inoculation casO and cas3 into 24 tubes
Notes: casO#1 goes to 1-6, casO#2 goes to 7-12; cas3#1 goes to another 1-6, cas3#2 goes to another 7-12
09/23/2011
1. Mini-prep all the casO and cas3 samples
2. Digest them by different enzymes
3. Ligate them with pSB1C3 vectors
09/24/2011
1. Transform all the ligated products into competent cells
2. Inoculate them in LB media
09/25/2011
1. Mini-prep the biobrick products
2. Verify them by PCR
09/25/2011
1. Transform
the casO plasmid into BL21; cas3; tetO::GFP; CRISPR-GFP
2. Inoculate the above plasmids
09/27/2011
1. Do growth test for the following BL21 cells:
BL21 tetO::GFP-IPTG
BL21 tetO::GFP+IPTG
BL21 tetO::GFP+IPTG(2 hours later)
BL21 tetO::GFP, CRISPR::GFP-IPTG
BL21 tetO::GFP, CRISPR::GFP+IPTG
BL21 tetO::GFP, CRISPR::GFP+IPTG(2 hours later)
BL21 tetO::GFP, CRISPR::GFP+cas3-IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+IPTG(2 hours later)
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E-IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E+IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E+IPTG(2 hours later)
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2-IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2+IPTG
BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2+IPTG(2 hours later)
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