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:

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.

Day of lab:

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!)

Recipes/Reagents

Growth media

1. 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.
2. 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.
3. Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.
4. Tet: 20 mg/ml MeOH. Keep in dark at 4°. Use at 1:1000 in liq media. 1ml/L in plates
5. 20% PEG-8000/2.5M NaCl (salt dissolved in PEG solution) Store at 4°.