20.109(S16): TA notes for module 3
Be sure to read the experimental protocols, materials, and timeline developed in Fall 2015 by Dr. Maryam Moradi and George Sun from the Belcher lab.
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:
In 4 °C fridge:
- orbital shaker (speed 120)
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.
- Returned 90 mL solution of phage-Fe+phosphate solution which was completed between Day 1 & 2 by instructors.
- 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.
- Flat stainless steel plate (#9785K241)
- 6" x 6"
- Thickness: 0.060"
- Tolerance: ±0.004"
- Smooth stainless steel roller (#1256T29)
- Dia: 3/4"
- Tolerance ±0.010"
- Length: 1 ft
- Stainless steel spatulas
- Pestle (#43095T62) and Mortar (#43095T52)
- Mortar OD: 3 1/8
- 3x 3/8 puncher
- superP carbon powder
- Teflon 8A (aka. binder)
- Ten groups were successfully handled between 2 TAs and 1 Instructor. The groups were split between rooms 76-589 and 76-591 of the Belcher lab. Each room has its own weigher and stock of superP and binder.
- Due to limited weighers, groups have to take turns weighing out their dried active material.
- Weights should be taken on a triangle cut weigh paper. Once weights are calculated, active material can be placed directly into the pestle
- Each group weighs out a calculated amount of superP and mixes it with their active material in the pestle.
- Team members should split up
- One should continue grinding their material. When complete (ask instructor/TA), gently transfer onto steel plate. DO NOT ROLL
- Another student should weigh out binder. Careful: small amounts (10-50 mg) of binder needs to be weighed, an error of +/- 10% is OK.
- Slowly and carefully transfer binder on its weigh paper and sprinkle on top of the mixed active material and superP
- Go crazy and start rolling
- Ask instructors for help or input if film is breaking, not shiny, or is difficult to punch out with the 3/8 puncher.
- All students electrodes should be weighed out and transferred gently into one well of a 6 well plate. The cover over each well should be clearly labeled with lab time and group color.
- TA: when a plate has an electrode in each well, put on cover and tape each edge. Transfer the plate into the 80 oC vacuum oven.
- Stainless steel plates, spatulas, and rollers can be cleaned with thorough washes of water to remove residual electrode materials.
- Cleaning the Pestle and Mortar is difficult. Clean with alternating washes of water and alconox. Many residual material may remain on the pestle, this is unavoidable. Pestle and Mortar can be further heated at > 8O oC overnight to etch away material that can be later washed away.
to the TEM! (CMSE link)
FEI Tecnai Multipurpose TEM
The FEI Tecnai (G2 Spirit TWIN) is a high quality 120 kV multipurpose TEM, providing high resolution and good contrast. The high-resolution TWIN lens allows for imaging at both moderate and high magnification up to 0.36 nm point-to-point resolution & 0.2 nm lattice resolution. The digital TEM combines excellent performance with optimized ease-of-use for high resolution imaging, elemental analysis and mapping.
- TEM needs to be reserved 2 - 3 weeks in advance! Email: email@example.com for permission with date and time.
- A TA and instructor should take 1/2 of the students to image their samples under TEM
- at the TEM, while imaging the last sample of that patch of students, send off the instructor with the remainder of the students back to the 20.109 lab. Instructor should then pick up the remaining students and bring them over to the TEM.
- Show students how to measure crystal dimensions from EM images (from Day 4)
- Download imageJ from the NIH website (google it)
- Open an EM image, usually a '.tif' file
- Mention that '.tif' files are not canonical image formats. This may lead to conflicts when embedding in word.doc, emails, or Evernote. '.tiff', '.png', '.bmp' are universal image extensions. '.png' has good quality versus data size. Use save as to save the '.tif' file format to something else.
- Click on the line icon on the menu bar
- Hold shift and drag a line across the scale bar.
- Go to Analyze > Set Scale. Fill in the necessary boxes, such as the actual dimension of the lined scale bar. Leave the width option as is.
- Now on, each line you draw is calibrated to the expected length established by the image's scale bar. Each image has to undergo its own scale calibration if the scale bars are different, or if the image is of a different pixel dimension.
- Draw a line across a crystal of interest. On the keyboard, push ctrl+M. This will open a table with parameters on the column and values on each row. Note that there are superfluous parameters, such as grey-scale, min/max, etc.
- To remove superfluous parameters, go to Analyze > Set Measurement and select any parameters you are interested in calculating. In our case, we need none of them, so we can deselect all of them
- Continue to draw lines and ctrl+M'ing. The table will populate with each line measurements.
- Highlight the table and copy+paste into an excel spreadsheet to calculate statistics.
- For overly ambitious students, students can use the Freehand selection icon to draw user-defined shapes. These shapes can be used to calculate area of crystals using the ctrl+M
- Mention the central limit theorem (CLT), most observations will behave a normal distribution with a defined mean and standard deviation. 33 or more observations are needed to converge an arbitrary data set to its nominal mean and std.