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

Biomaterials Engineering Module

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General notes

Before term/module begins:

1. Check that all links on all wiki pages for biomaterial engineering modulre properly direct to current version of class and to working webpages. Fix broken ones.
2. Dissolve 1.7 g Ir(III)Cl3 in 200 ml ddH20. Do not use IrCl3 from Sigma (cat # 336807) since this is NOT water soluble (oddly). Use only Alfa Aesar IrCl3 (Cat # 11030). Store in dark. note for F07 this has already been done. Most all of the materials for the module are in one cabinet in the main lab. Check there first.
3. Confirm pH meter is in good working order.
4. 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.
5. Wake up Blattner minimal strains on LB plate at 37° ON. These are NB258 through NB263.
6. Wake up M13 titerin strain, NB263 = 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'.
7. Pour LB plates (4L), 4 or 6 plates per group, x 2 days.
8. Prepare top agar if needed.
9. Autoclave more large and small tubes if necessary.
10. Check stock of LB liquid media and make more if necessary.
11. Reserve TEM for two afternoons it will be needed if this has not been done. Arrange for operator of TEM in you are not certified.

Notes: 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 titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group.

Day of lab:

1. Need quiz (all quizzes 2 or 3 questions, 5 points).
2. Aliquot PEG solution for each group.
3. Place rack of 15 ml falcon tubes and foil out for Ir pH'ing.
4. Distribute LB plates for titering phage. Need at least 4/lab group.
5. Melt top agar. Need to melt 15 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.

Notes: Day 2

Before lab:

Day of lab:

1. Need quiz

Notes:Day 3

Before lab:

1. Make 2L 1X TAE for gels and running buffer
2. Pour 3 agarose gels, 1% in TAE (100ml) + 2 ul EtBr each. Use 2 10-tooth combs/gel.
3. Aliquot solutions (see below)

Day of lab:

1. Need quiz
2. Aliquot solutions for miniprep (Solutions 1, 3, NaOH and SDS) for each group so stocks don't get contaminated.
   • Each group needs:
     • 400 ul Solution 1 (6 microtubes of 1000 ul)
     • 500 ul SDS 2% (6 microtubes of 1000 ul)
     • 500 ul 0.4M NaOH (6 microtubes of 1000 ul)
     • 600 ul Sol 3 (6 microtubes of 1000 ul)
     • 4 ml 100% Ethanol (6 x full 15 ml tubes)
     • 2 ml 70% Ethanol (6 x ~10 ml in 15 ml tubes)
     • sterile water bottle for each group
3. Keep cold:
   • buffers
   • restriction enzymes
4. ice buckets (one per bench should be fine, just for miniprep)
5. 1 ml serological pipets for alcohols (optional)
6. loading dye for gels

Notes:Day 6

Day before lab:

1. Start overnights of 7x NB271 liquid cultures for plaque assay (3 ml of LB+Kan in large tubes), each group needs ~2 ml cells.
2. Start overnights of 7x NB251 liquid cultures for + control on Western (3 ml of LB+Kan in large tubes), each group needs ~1 ml.
3. Pour 2L of LB plates
4. Have protein gels and chambers
5. For each day, make microtubes of 1X sample buffer without BME (500 ul 2X + 400 ul H20) add 100ul of BME to both on day of lab (could parafilm after adding BME, save 2 days). Need <1 ml / group.
6. Make 3 L of transfer buffer for each day and refrigerate
7. Make 1L 1X TBS-T in graduated cylinder, refrigerate, mix in 5% powdered milk close to lab day

• Note: 2°Ab is goat antimouse (GAM-AP) in ab freezer box

Day of lab:

• Need quiz
• Turn on water baths and melt top agar
• Protein gels and blot

1. Students' candidates in liquid culture tubes
2. Blank for spec (900 ul H2O and 100 ul LB)
3. Sample buffer + 100 ul BME
4. Lid locks for microtubes in hood
5. Boiling tank on hot plate in hood with boiling chips, be sure it's turned off once students are done.
6. 2 protein gel chambers with 2 gels in one and 1 plus spacer in other, set up
7. "Kaleidoscope" protein molecular weight standard, in -20C in enzyme rack, aliquot 2x50 ul in microtubes, denature by boiling when students boil their samples
8. Running buffer, 1X TGS made when needed, 1L/chamber, 10X above sink
9. Transfer cassettes, ScotchBrite pads, filter paper, nitrocellulose
10. Western transfer buffer, 1L/tank so 3L/day
11. ice packs for Western
12. Blocking buffer TBS-T + 5% milk, 50 ml/group
13. Protein gels will have two groups/gel so that blot can be cut in half, and each probed with anti-p3.

• Plaque assay

1. top agar, melted in microwave and kept in 55C water bath
2. 200 ul x 10 /group NB271(=ER2267) bacteria for plaque assay
3. 10^6 dilution of positive phage control in microtube
4. small sterile test tubes, 10/group
5. 10 LB plates/group for plaque assay (no phage, + phage control, 4 dilutions supernatant: 10^0,2,4,6, for each of two candidates)
6. 5 ml pipets and Pipetmen

Notes:Day 7

Before lab:

Day of lab:

Recipes/Reagents

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°.
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. 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°.