Team:USC/Notebook/Week10

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Revision as of 06:09, 25 September 2011

Official Team Profile

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		+		+		+

06/11/2011
1. Purify plasmid DNA from the culture

@@ Line 196: / Line 196: @@
 reaction mixture as follows:
 <br />
-<table>
+<table border="1">
 <tr>
 <td></td><td>A</td><td>B</td><td>C</td><td>D</td><td>E</td><td>F</td>
@@ Line 203: / Line 203: @@
 <td>Amp</td><td>+</td> <td>+</td> <td> </td> <td> </td> <td>+</td> <td>+</td>
 </tr>
+<tr>
+<td>CM</td><td></td> <td></td> <td>+</td> <td>+</td> <td>+</td> <td>+</td>
+</tr>
+<tr>
+<td>IPTG</td><td></td> <td>+</td> <td></td> <td>+</td> <td></td> <td>+</td>
+</tr>
 </table>