"
Team:Caltech/Protocols
From 2011.igem.org
| Line 3: | Line 3: | ||
[[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Recipes|Recipes for Mixes]]<br/><br/> | [[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Recipes|Recipes for Mixes]]<br/><br/> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<p>'''Enrichment cultures'''<br/> | <p>'''Enrichment cultures'''<br/> | ||
| - | |||
* For BPA (since soluble)<br/> | * For BPA (since soluble)<br/> | ||
1. Set up 16 tubes: 8 tubes with vitamin media vs. 8 tubes with media (no vitamin), 4 tubes for each of the four locations.<br/> | 1. Set up 16 tubes: 8 tubes with vitamin media vs. 8 tubes with media (no vitamin), 4 tubes for each of the four locations.<br/> | ||
| Line 23: | Line 12: | ||
2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.<br/> | 2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.<br/> | ||
For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.<br/><br/></p> | For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.<br/><br/></p> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
<p>'''Gibson Assembly (Adapted from Cambridge 2010)'''<br/> | <p>'''Gibson Assembly (Adapted from Cambridge 2010)'''<br/> | ||
| Line 37: | Line 19: | ||
2. To 3uL of DNA, add 7.5uL of Gibson Mix<br/> | 2. To 3uL of DNA, add 7.5uL of Gibson Mix<br/> | ||
3. Incubate @ 50C for 30-60 minutes with heated lid<br/> | 3. Incubate @ 50C for 30-60 minutes with heated lid<br/> | ||
| - | 4. Cool, then transform into chemically competent cells<br/></p> | + | 4. Cool, then transform into chemically competent cells<br/></p><br/> |
| + | |||
| + | <p>'''Mobio PowerMax Soil kit''': http://www.mobio.com/images/custom/file/protocol/12988-10.pdf</p><br/> | ||
| + | |||
| + | <p>'''Pulse Gel Field Electrophoresis''':<br/> | ||
| + | PFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE<br/> | ||
| + | Parameters: 6 V/cm, 15°C for 20 hours.<br/> | ||
| + | Switch times ramped from 0.5-1.5 seconds.</p><br/> | ||
| + | |||
| + | <p>'''Qiagen Miniprep kit''': www.qiagen.com/hb/qiaprepminiprep<br/></p><br/> | ||
| + | |||
| + | <p>'''Transforming DNA from Distribution Plates:'''<br/> | ||
| + | 1. Thaw competent cells on ice.<br/> | ||
| + | 2. Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.<br/> | ||
| + | 3. Transfer into storage tube.<br/> | ||
| + | 4. Pipette 1-2 microliters of the DNA into the competent cell tubes.<br/> | ||
| + | 5. Stir with pipette tip, gently flick tube.<br/> | ||
| + | 6. Leave on ice for 30 minutes.<br/> | ||
| + | 7. Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.<br/> | ||
| + | 8. Pipette 500 micro Liters of S.O.C. (LB + glucose) into 14ml culture tubes; transfer the competent cells into these tubes and incubate in a 37 degree shaker for 0-60 minutes before plating.<br/> | ||
| + | 9. For source plate DNA, plate 100 microliters.</p><br/> | ||
}} | }} | ||
Revision as of 22:41, 14 July 2011
|
Project |
Back to Timeline . Recipes for Mixes Enrichment cultures
2. Place 8 test tubes in 30°C shaker and 8 test tubes in room temperature shaker.
2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask. For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures. Gibson Assembly (Adapted from Cambridge 2010) Mobio PowerMax Soil kit: http://www.mobio.com/images/custom/file/protocol/12988-10.pdf Pulse Gel Field Electrophoresis: Qiagen Miniprep kit: www.qiagen.com/hb/qiaprepminiprep Transforming DNA from Distribution Plates:
|
