Difference between revisions of "20.109(F15): TA's notes for module 3"

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(→‎Day 2)
(→‎Day 2)
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'''Between Day 1 and Day 2:'''
 
'''Between Day 1 and Day 2:'''
 
*1 M pH 7.5 Na:PO<sub>4</sub> solution:
 
*1 M pH 7.5 Na:PO<sub>4</sub> solution:
−
** 2.6% Monosodium phosphate, monohydrate - 2.6 g per 100 mL
+
**2.6% Monosodium phosphate, monohydrate - 2.6 g per 100 mL
−
** 21.8% Disodium phosphate, heptahydrate - 21.8 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
+
**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 <br>
 +
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)
  
Line 51: Line 53:
  
 
'''On students' benches:'''
 
'''On students' benches:'''
−
*Na:PO<sub>4</sub>
+
*For demo purposes:
−
*M13 phage with(NH<sub>4</sub>)<sub>2</sub>Fe(SO<sub>4</sub>)<sub>2</sub>
+
** ~10 mL of each students phage-Fe mixture which was saved previously
−
quantities to be updated by George
+
** 1 mL aliquot of 1 M pH 7.5 Na:PO<sub>4</sub> solution in eppendorf
 +
** Allow students to add 100 &mu;L 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 Fe<sub>2</sub>O<sub>3</sub>) 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.

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.
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