20.109(F07): TA's notes for module 3

Biomaterials Engineering Module
20.109(F08) 20.109(F07) (archive)

General notes
Before module begins:
 * 1) 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.
 * 2) Reserve TEM for Day 5
 * 3) 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.
 * 4) 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'.
 * 5) Pour LB plates
 * 6) Pour LB+ Kan plates
 * 7) Prepare top agar if needed.
 * 8) Autoclave more large and small glass tubes if necessary.
 * 9) Check stock of LB liquid media and make more if necessary.

Day 1
Before lab: Day of lab:
 * 1) Grow phage infected strain NB273, enough for each pair of students to work with at least 3 ml.
 * 2) Check pH meter area for water, waste beaker, standards, Chemwipes, pasteurs, bulbs, pH paper etc.
 * 3) Grow NB271= titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group.
 * 1) Do not need quiz on day 1 but in future all quizzes 2 or 3 questions, 5 points.
 * 2) Aliquot PEG solution for each group. (500 ul/group in eppendorfs should be enough)
 * 3) Fill ice buckets for each group
 * 4) Place rack of 15 ml falcon tubes and foil out for Ir pH'ing.
 * 5) Place rack of small sterile test tubes out where all groups can collect as needed.
 * 6) Place 5 ml pipets out near 55° water bath with melted top agar.
 * 7) 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.
 * 8) 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:
 * 1) Remove petri dishes from 37° incubator and store at 4° or RT until next time.

Day 2
Before lab:

Day of lab:
 * 1) Need quiz
 * 2) 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:
 * 1) Confirm reservation for TEM

Day of lab:
 * 1) 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!)

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.
 * 1) 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.
 * 2) Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.
 * 3) Tet: 20 mg/ml MeOH. Keep in dark at 4°. Use at 1:1000 in liq media. 1ml/L in plates
 * 4) 20% PEG-8000/2.5M NaCl (salt dissolved in PEG solution) Store at 4°.