20.109(F07): TA's notes for module 3
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Biomaterials Engineering Module
20.109(F08)
20.109(F07) (archive)
General notes
Before module begins:
- Check that all links on all wiki pages for biomaterial engineering module properly direct to current version of class and to working webpages. Fix broken ones.
- Reserve TEM for Day 5
- Wake up NB273, strain with 3-12 version of M13 in it.Since phage has no selectible marker try to recover by innoculating 2 ml of LB+tet with dab from frozen stock. Tet selects for F' in host strain.
- Wake up M13 titering strain, NB271 = ER2267 cat#E4103S. Streak out a plate on LB+Kan so you will have colonies to pick for liquid cultures before phage titration. The Kan is important to select for the F'.
- Pour LB plates
- Pour LB+ Kan plates
- Prepare top agar if needed.
- Autoclave more large and small glass tubes if necessary.
- Check stock of LB liquid media and make more if necessary.
Day 1
Before lab:
- Grow phage infected strain NB273, enough for each pair of students to work with at least 3 ml.
- Check pH meter area for water, waste beaker, standards, Chemwipes, pasteurs, bulbs, pH paper etc.
- Grow NB271= titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group.
Day of lab:
- Do not need quiz on day 1 but in future all quizzes 2 or 3 questions, 5 points.
- Aliquot PEG solution for each group. (500 ul/group in eppendorfs should be enough)
- Fill ice buckets for each group
- Place rack of 15 ml falcon tubes and foil out for Ir pH'ing.
- Place rack of small sterile test tubes out where all groups can collect as needed.
- Place 5 ml pipets out near 55° water bath with melted top agar.
- Place LB plates at RT for titering phage. Need at least 6/lab group. Can leave as piles of plates at front of room where groups can collect as needed.
- Melt top agar. Need to melt so at least 20 ml of top agar/group. Microwave 1', swirl, microwave 30", and then into 55C water bath to keep molten. It's very important that the top agar be fully melted for the students.
Day after lab:
- Remove petri dishes from 37° incubator and store at 4° or RT until next time.
Day 2
Before lab:
Day of lab:
- Need quiz
- Distribute titer plates from Day 1
Day after lab:
Day 3
- no prep since today is Journal Club (yay!)
Day 4
Before lab:
Day of lab:
- need quiz
At end of lab:
Day after lab:
Day 5
Day before lab:
- Confirm reservation for TEM
Day of lab:
- Need quiz.
Day after lab:
Day 6
Before lab:
Day of lab:
Day 7
- no lab prep since this day is for presentations of research ideas (yay!)
Recipes/Reagents
Growth media
- LB: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter. 20g of Agar for plates. Autoclave 30 minutes with stirbar. Pour when ~55°. Let plates dry ON on bench and store in sleeves in 4°. For LB-Kan plates, add the Kan after autoclaving, once the mixture has cooled down to a temperature where you can hold your hands on the flask and not feel like you're being burned.
- Top Agar: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, 1 g MgCl2*6H20 7 g Agar per liter. Autoclave then aliquot to 50 ml conical tubes or bottles. Store at RT. Melt in microwave in beaker of water, 2’ then keep molten in 55° water bath.
- Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.
- Tet: 20 mg/ml MeOH. Keep in dark at 4°. Use at 1:1000 in liq media. 1ml/L in plates
- 20% PEG-8000/2.5M NaCl (salt dissolved in PEG solution) Store at 4°.