20.109(F09): TA's notes for module 3: Difference between revisions
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= | '''Friday November 6''' | ||
* | *Make up overnight inoculum of phage for Thursday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight | ||
* | *autoclave/sterilize 16 125-mL flasks (one flask per group) | ||
* | *make sure there are enough Oak Ridge tubes for each group (=3 per group) | ||
* | *make sure that there is a centrifuge available for Thursday and Friday afternoon for the PEG precipitation (Niles lab down the hall?) | ||
''' | *make enough PEG solution (20% PEG-8000, 2.5 M NaCl --- this requires a long time to fully solubilize - leave on the stir plate for about an hour) | ||
*Each group should make a 0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB"). This is done by mixing 0.72 g with 20 ml of water in a 50 ml conical tube. | |||
*One group will be responsible for making each of the following solutions that the class will use in the synthesis of nanowires. | '''Saturday November 7''' | ||
* | *Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks (for Thursday class). Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Monday night) | ||
**10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O) = Mix 40 mg with 10 mL water | |||
**10 mM silver nitrate (AgNO3)= Mix 17 mg with 10 mL water | |||
** 0.1 M ascorbic acid = Mix 0.18 gram with 10 mL water | '''Sunday November 8''' | ||
** 2.5 M NaCl = Mix 7.3 g NaCl with 50 ml water | *Make up overnight inoculum of phage for Friday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight | ||
'''Monday November 9''' | |||
*Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Thursday's class) | |||
*Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks. Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Wednesday night) | |||
'''Tuesday November 10''' | |||
*check to make sure there are enough LB plates for each group to have 4 total | |||
*check to see that there is enough top agar for each group to have 12 mL total | |||
'''Wednesday November 11''' | |||
*Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Friday's class) | |||
*Use small (yellow-capped) tubes to make titering cells - about 1 mL is necessary for each group, so make up 6 5-mL cultures with 5 mL LB, 5 µL Kan, and a colony of ER2267. Incubate rotating overnight at 37ºC | |||
'''Set up for Day 1''' | |||
*ice buckets (three) filled with ice for PEG precipitation | |||
*40 mL phage cultures | |||
*titering cells (ER2267) | |||
*necessary chemicals (CTAB, hydrated hydrogen tetrachloroaurate, silver nitrate, ascorbic acid, and NaCl) | |||
**Each group should make a 0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB"). This is done by mixing 0.72 g with 20 ml of water in a 50 ml conical tube. | |||
**One group will be responsible for making each of the following solutions that the class will use in the synthesis of nanowires. | |||
***10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O) = Mix 40 mg with 10 mL water | |||
***10 mM silver nitrate (AgNO3)= Mix 17 mg with 10 mL water | |||
***0.1 M ascorbic acid = Mix 0.18 gram with 10 mL water | |||
***2.5 M NaCl = Mix 7.3 g NaCl with 50 ml water | |||
'''Set up for Day 2''' | |||
*quiz! | |||
'''Other important notes:''' | |||
*all antibiotic solutions are stored at 4ºC but powdered tet is in the -20ºC and powdered kan is in the 4ºC | |||
*ER2267 cells are kind of picky and die after some time at 4ºC - streak out a new plate from the stock at -80ºC before beginning the module |
Revision as of 08:25, 11 November 2009
Friday November 6
- Make up overnight inoculum of phage for Thursday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight
- autoclave/sterilize 16 125-mL flasks (one flask per group)
- make sure there are enough Oak Ridge tubes for each group (=3 per group)
- make sure that there is a centrifuge available for Thursday and Friday afternoon for the PEG precipitation (Niles lab down the hall?)
- make enough PEG solution (20% PEG-8000, 2.5 M NaCl --- this requires a long time to fully solubilize - leave on the stir plate for about an hour)
Saturday November 7
- Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks (for Thursday class). Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Monday night)
Sunday November 8
- Make up overnight inoculum of phage for Friday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight
Monday November 9
- Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Thursday's class)
- Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks. Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Wednesday night)
Tuesday November 10
- check to make sure there are enough LB plates for each group to have 4 total
- check to see that there is enough top agar for each group to have 12 mL total
Wednesday November 11
- Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Friday's class)
- Use small (yellow-capped) tubes to make titering cells - about 1 mL is necessary for each group, so make up 6 5-mL cultures with 5 mL LB, 5 µL Kan, and a colony of ER2267. Incubate rotating overnight at 37ºC
Set up for Day 1
- ice buckets (three) filled with ice for PEG precipitation
- 40 mL phage cultures
- titering cells (ER2267)
- necessary chemicals (CTAB, hydrated hydrogen tetrachloroaurate, silver nitrate, ascorbic acid, and NaCl)
- Each group should make a 0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB"). This is done by mixing 0.72 g with 20 ml of water in a 50 ml conical tube.
- One group will be responsible for making each of the following solutions that the class will use in the synthesis of nanowires.
- 10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O) = Mix 40 mg with 10 mL water
- 10 mM silver nitrate (AgNO3)= Mix 17 mg with 10 mL water
- 0.1 M ascorbic acid = Mix 0.18 gram with 10 mL water
- 2.5 M NaCl = Mix 7.3 g NaCl with 50 ml water
Set up for Day 2
- quiz!
Other important notes:
- all antibiotic solutions are stored at 4ºC but powdered tet is in the -20ºC and powdered kan is in the 4ºC
- ER2267 cells are kind of picky and die after some time at 4ºC - streak out a new plate from the stock at -80ºC before beginning the module