Difference between revisions of "20.109(F15): TA's notes for module 3"
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**These are calculated values that will result in a 100 mL solution of pH 7.5 | **These are calculated values that will result in a 100 mL solution of pH 7.5 | ||
| − | *Aliquot ~ 10 mL of students phage-Fe incubating mixture and transfer to 20 mL glass vial | + | *Aliquot ~ 10 mL of students phage-Fe incubating mixture and transfer to 20 mL glass vial |
This will be used for demonstration purposes for their next lab section | This will be used for demonstration purposes for their next lab section | ||
*12-16 hours after phage-Fe incubation, add 1 mL of Na:PO<sub>4</sub> solution to each groups flask (result is a 10 mM phosphate solution) | *12-16 hours after phage-Fe incubation, add 1 mL of Na:PO<sub>4</sub> solution to each groups flask (result is a 10 mM phosphate solution) | ||
Revision as of 02:33, 18 November 2015
Day 1
The week preceding the lab:
- Make LB:
- 10 g tryptone
- 5 g yeast extract
- 10 g NaCl
- 1 L deionized water
- autoclave 30 minutes
On the instructors' bench:
- 50 mL conical tubes
- 25 mL pipets
- a couple of motorized pipet aids
- 2 mL eppendorf tubes
- TBS: 500 mL
- PEG/NaCl: 500 mL
- deionized water: 50 mL
- quartz cuvettes
On the students' bench:"
- bucket of ice
- 2 x 40 mL overnight culture of XL1-Blue infected with M13 phage
- 250 mL erlenmeyer flask
By the scale:
- (NH4)2Fe(SO4)2
- stirbars
In 4 °C fridge:
- orbital shaker (speed 120)
Day 2
Between Day 1 and Day 2:
- 1 M pH 7.5 Na:PO4 solution:
- 2.6% Monosodium phosphate, monohydrate - 2.6 g per 100 mL
- 21.8% Disodium phosphate, heptahydrate - 21.8 g per 100 mL
- These are calculated values that will result in a 100 mL solution of pH 7.5
- Aliquot ~ 10 mL of students phage-Fe incubating mixture and transfer to 20 mL glass vial
This will be used for demonstration purposes for their next lab section
- 12-16 hours after phage-Fe incubation, add 1 mL of Na:PO4 solution to each groups flask (result is a 10 mM phosphate solution)
On the instructors' bench:
- 50 mL conical tubes
- 15 mL conical tubes
- tweezers (2 per team)
- TEM grids for practice (some)
- new TEM grids (2 per team)
- sign-up sheet for location of samples in TEM holder
- 100% ethanol (20 μL per team, so a couple of 1.5 mL eppendorf tubes suffice)
On students' benches:
- For demo purposes:
- ~10 mL of each students phage-Fe mixture which was saved previously
- 1 mL aliquot of 1 M pH 7.5 Na:PO4 solution in eppendorf
- Allow students to add 100 μL of phosphate solution to their phage-Fe mixture
- Solution should turn green-ish brown. The darker color, compared to their overnight phage-Fe+phosphate mixture may be due to the prolonged phage-Fe incubation (which lasted over the weekend), which may have formed iron oxide particles (FeO or Fe2O3) that are more opaque and rusty colored.
Note:
- An instructor should go around with the grid holder, and allow each group to take two tweezers and grab two Cu-TEM grids
- Grids should be held by the tweezers at all times during depositing and washing steps.
- Place some kimwipes beneath the tweezers, and set tweezers with grid on the bench.
