Team:USC/Notebook/Week13

From 2011.igem.org

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3. Make HT115(CRISPR-GFP #7) competent and make freeze stock
3. Make HT115(CRISPR-GFP #7) competent and make freeze stock
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<tr>
 
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<td colspan="7">
 
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<h3 style="font-family:Verdana; font-weight:700;background-color: #F0F0F0;">'''Week 14:'''</h3>
 
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<span style="font-family:Verdana; font-weight:700;"> 09/13/2011 </span>
 
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<br />
 
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1. Check quality of gDNA
 
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<br />
 
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2. Make HT115 + BL21 (tetO, CRISPR-GFP #7) competent cells, freeze stock
 
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<br />
 
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3. Miniprep PCDF+casO
 
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<br />
 
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C. Plate 4 Well 16O  (LovTAP Composite)
 
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<br />
 
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4. Digest tetO/tetR
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/15/2011 </span>
 
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1. Run a gel of all minipreped PCDF+casO<br />
 
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<br />
 
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2. PCR HT115, DH5α2, DH5α3 with primers<br />
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/18/2011 </span>
 
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<br />
 
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1. Inoculate the following:
 
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<br />
 
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BL21 tetO::GFP
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7
 
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BL21 tetO::GFP, CRISPR::GFP#7, cas3
 
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<br />
 
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HT115 tetO::GFP
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7, cas3
 
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<br />
 
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HT115 CRISPR::GFP #7
 
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<span style="font-family:Verdana; font-weight:700;"> 09/19/2011 </span>
 
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1. Growth test, grow all the bacteria overnight<br />
 
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<br />
 
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2. PCR cas3+casO from gDNA
 
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<span style="font-family:Verdana; font-weight:700;"> 09/20/2011 </span>
 
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1.      Make biobrick of cas3 and casO
 
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<br />
 
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2. Run a gel for cas3 and casO
 
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<br />
 
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3. Purify cas3 and casO
 
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<br />
 
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4. New experiment: dilution the bacteria to 1000 and 5000 cells and plate them
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/21/2011 </span>
 
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<br />
 
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1.      Do growth test for the following:
 
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<br />
 
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BL21 tetO::GFP-IPTG
 
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<br />
 
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BL21 tetO::GFP+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7, cas3-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7, cas3+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7, cas3+IPTG an hour later
 
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<br />
 
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HT115 tetO::GFP-IPTG
 
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<br />
 
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HT115 tetO::GFP+IPTG
 
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<br />
 
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HT115 tetO::GFP+IPTG an hour later
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7-IPTG
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7+IPTG
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7+IPTG an hour later
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7, cas3-IPTG
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7, cas3+IPTG
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7, cas3+IPTG an hour later
 
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<br />
 
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HT115 CRISPR::GFP #7-IPTG
 
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<br />
 
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HT115 CRISPR::GFP #7+IPTG
 
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<br />
 
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HT115 CRISPR::GFP #7+IPTG an hour  later
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP#7, cas3
 
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<br />
 
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HT115 tetO::GFP
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7
 
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<br />
 
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HT115 tetO::GFP, CRISPR::GFP#7, cas3
 
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<br />
 
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HT115 CRISPR::GFP #7
 
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<br />
 
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2.      Dilution: plate on Amp #4 and #5 +/- IPTG, #4 and #5 have different OD values. Dilute 1000* and 5000* to both of them
 
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<br />
 
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3.      Digest pSB1C3 and tetO/R vector
 
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<br />
 
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4.      Transform casO#1, #2, #1, #2 on CM plates
 
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<span style="font-family:Verdana; font-weight:700;"> 09/22/2011 </span>
 
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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
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/23/2011 </span>
 
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<br />
 
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1. Mini-prep all the casO and cas3 samples
 
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<br />
 
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2. Digest them by different enzymes
 
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<br />
 
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3. Ligate them with pSB1C3 vectors
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/24/2011 </span>
 
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<br />
 
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1. Transform all the ligated products into competent cells
 
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<br />
 
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2. Inoculate them in LB media
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/25/2011 </span>
 
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<br />
 
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1. Mini-prep the biobrick products
 
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<br />
 
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2. Verify them by PCR
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/25/2011 </span>
 
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<br />
 
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1. Transform
 
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the casO plasmid into BL21; cas3; tetO::GFP; CRISPR-GFP
 
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<br />
 
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2. Inoculate the above plasmids
 
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<br />
 
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<span style="font-family:Verdana; font-weight:700;"> 09/27/2011 </span>
 
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<br />
 
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1.      Do growth test for the following BL21 cells:
 
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<br />
 
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BL21 tetO::GFP-IPTG
 
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<br />
 
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BL21 tetO::GFP+IPTG
 
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<br />
 
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BL21 tetO::GFP+IPTG(2 hours later)
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+IPTG(2 hours later)
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+IPTG(2 hours later)
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E+IPTG
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-E+IPTG(2 hours later)
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2-IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2+IPTG
 
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<br />
 
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BL21 tetO::GFP, CRISPR::GFP+cas3+casA-2+IPTG(2 hours later)
 
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<br />
 
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</table>
 

Revision as of 03:36, 29 September 2011

USC-logo.jpg IGEM2011-logo.jpg IGEM-logo.jpg
USC Banner.jpg

Brainstorming
Brain storm.JPG

Week 1
Week1.jpg

Week 2
Week2.jpg

Week 3
Week3.jpg

Week 4
Week4.jpg

Week 5
Week5.jpg

Week 6
Week6.jpg

Week 7
Week7.jpg

Week 8
Week8.jpg

Week 9
Week9.jpg

Week 10
Week10.jpg

Week 11
Week11.jpg

Week 12
Week12.jpg

Week 13
Week13.jpg

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


Retrieved from "http://2011.igem.org/Team:USC/Notebook/Week13"