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Team:USC/Notebook/Week10
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| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC|Home]]</span></span> |
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| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Project|Project]]</span></span> |
| - | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Notebook|Notebook]]</span></span> | |
| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Parts|Parts]]</span></span> |
| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Safety|Safety]]</span></span> |
| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Human Outreach|Human Outreach]]</span></span> |
| - | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: | + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[[Team:USC/Attributions|Attributions]]</span></span> |
| + | <span style="float: left; padding: 15px;"><span style="font-family: Arial, Helvetica, sans-serif;font-size: 15px;font-weight: bold;color: #FFFFFF;border: none;">[https://igem.org/Team.cgi?year=2011&team_name=USC Official Team Profile]</span></span> | ||
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<table> | <table> | ||
<tr> | <tr> | ||
| + | <h1 style="font-family:Verdana;font-weight:700;">Laboratory Notebook</h1> | ||
| + | </tr> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
[[Team:USC/Notebook/Brainstorming|Brainstorming]] | [[Team:USC/Notebook/Brainstorming|Brainstorming]] | ||
<br/> | <br/> | ||
| - | [[File:Brain_storm.JPG]] | + | [[File:Brain_storm.JPG|link=Team:USC/Notebook/Brainstorming|Brainstorming]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week1|Week 1]] |
<br/> | <br/> | ||
| - | [[File:week1.jpg]] | + | [[File:week1.jpg|link=Team:USC/Notebook/Week1|Week 1]] |
</td> | </td> | ||
| - | + | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | |
| + | [[Team:USC/Notebook/Week2|Week 2]] | ||
| + | <br/> | ||
| + | [[File:week2.jpg|link=Team:USC/Notebook/Week2|Week 2]] | ||
| + | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week3|Week 3]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week3.jpg|link=Team:USC/Notebook/Week3|Week 3]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week4|Week 4]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week4.jpg|link=Team:USC/Notebook/Week4|Week 4]] |
</td> | </td> | ||
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| + | <tr> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week5|Week 5]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week5.jpg|link=Team:USC/Notebook/Week5|Week 5]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week6|Week 6]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week6.jpg|link=Team:USC/Notebook/Week6|Week 6]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week7|Week 7]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week7.jpg|link=Team:USC/Notebook/Week7|Week 7]] |
</td> | </td> | ||
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<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week8|Week 8]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week8.jpg|link=Team:USC/Notebook/Week8|Week 8]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week9|Week 9]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week9.jpg|link=Team:USC/Notebook/Week9|Week 9]] |
</td> | </td> | ||
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| + | <tr> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week10|Week 10]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week10.jpg|link=Team:USC/Notebook/Week10|Week 10]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week11|Week 11]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week11.jpg|link=Team:USC/Notebook/Week11|Week 11]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week12|Week 12]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week12.jpg|link=Team:USC/Notebook/Week12|Week 12]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week13|Week 13]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week13.jpg|link=Team:USC/Notebook/Week13|Week 13]] |
</td> | </td> | ||
<td style="text-align:center; font-family:Verdana; font-weight:700;"> | <td style="text-align:center; font-family:Verdana; font-weight:700;"> | ||
| - | [[Team:USC/Notebook/ | + | [[Team:USC/Notebook/Week14|Week 14]] |
<br/> | <br/> | ||
| - | [[File: | + | [[File:week14.jpg|link=Team:USC/Notebook/Week14|Week 14]] |
| + | </td> | ||
| + | |||
| + | </tr> | ||
| + | </table> | ||
</td> | </td> | ||
</tr> | </tr> | ||
| - | < | + | </table> |
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<h3 style="font-family:Verdana; font-weight:700;background-color: #F0F0F0;">'''Week 10:'''</h3> | <h3 style="font-family:Verdana; font-weight:700;background-color: #F0F0F0;">'''Week 10:'''</h3> | ||
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reaction mixture as follows: | reaction mixture as follows: | ||
<br /> | <br /> | ||
| - | <table> | + | <table border="1"> |
<tr> | <tr> | ||
| - | <td></td>< | + | <td></td><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>F</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| - | < | + | <th>Amp</th><td>+</td> <td>+</td> <td> </td> <td> </td> <td>+</td> <td>+</td> |
| + | </tr> | ||
| + | <tr> | ||
| + | <th>CM</th><td></td> <td></td> <td>+</td> <td>+</td> <td>+</td> <td>+</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <th>IPTG</th><td></td> <td>+</td> <td></td> <td>+</td> <td></td> <td>+</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
| + | <br /> | ||
| + | 2. Freeze down the leftover cultures in 4 tubes | ||
| + | <br/ > | ||
| - | <span style="font-family:Verdana; font-weight:700;"> | + | <span style="font-family:Verdana; font-weight:700;">08/19/2011 </span> |
<br /> | <br /> | ||
| - | 1. | + | 1. Take CRISPR+GFP with tetO::GFP competent cells, redilute, adding Am and CM, reaction mixture is as follows: |
| - | <br /> | + | <br/ > |
| - | </ | + | 5 µL culture+5mL LB+5 µL Am+5 µL CM |
| + | <br/ > | ||
| + | 5 µL culture+5mL LB+5 µL Am+5 µL CM+5 µL IPTG | ||
| + | <br/ > | ||
| + | Notes: add IPTG right away | ||
| + | <br/ > | ||
| + | 2. Mini-prep CASO+PCDF plasmid only with strep | ||
| + | <br/ > | ||
Latest revision as of 03:58, 29 September 2011
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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 | + | + | + |
2. Freeze down the leftover cultures in 4 tubes
08/19/2011
1. Take CRISPR+GFP with tetO::GFP competent cells, redilute, adding Am and CM, reaction mixture is as follows:
5 µL culture+5mL LB+5 µL Am+5 µL CM
5 µL culture+5mL LB+5 µL Am+5 µL CM+5 µL IPTG
Notes: add IPTG right away
2. Mini-prep CASO+PCDF plasmid only with strep
