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

Biomaterials Engineering Module

no wiki archive version

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. note for F07 this has already been piloted and worked well.
5. Wake up Blattner minimal strains on LB plate at 37° ON. These are NB258 through NB263. note for F07 this has already been done.
6. 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'.
7. Pour LB plates (4L), 6 plates per group, x 2 days.
8. Pour LB+ Kan plates (2L), 4 plates per group, x 2 days.
9. Prepare top agar if needed. TA left over from Mod 1 will still work as long as it's not contaminated.
10. Autoclave more large and small tubes if necessary.
11. Check stock of LB liquid media and make more if necessary.
12. 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. note for F07 this has already been done.

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. 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.

Notes: Day 2

Before lab:

Day of lab:

1. Need quiz
2. Distribute titer plates from Day 1
3. Place electropulser on bench with balance.
4. Place cuvettes where students can collect as needed.
5. Place nutator on bench across from balance with foil to cover.
6. Place dialysis tubing and clamps on teacher demo area.

Day after lab:

1. Change water in dialysis bath.

Notes: Day 3

Before lab:

Day of lab:

1. no quiz since students come to lab at staggered times to get to TEM at different times.
2. set up bench work area with tweezers, TEM grids, and petri dishes with Western blotting paper.

Notes: Day 4

Before lab:

1. Grow Blattner MDS strains, NB258 through NB263, in 2.5 ml LB 37° ON
2. Refill EtOH in burners and place EtOH lamps, strikers, jars of EtOH and spreaders on front bench

Day of lab:

1. Need quiz.
2. 5 hours before lab innoculate 0.5 ml into 4.5 ml LB of NB259 through NB263 and place on roller drum 37°
3. 2.5 hours before lab innoculate 8x 0.5 ml of NB258 into 4.5 ml LB and place on roller drum 37°
4. just before lab, fill ice buckets for all student benches.
5. aliquot 3 ml TB into 15 ml falcon tubes x6 (one aliquot/group)
6. aliquot 3x 0.5 ml DMSO into eppendorf tubes for front bench.
7. need 3 LB+Kan plates/group.
8. aliquot M13K07 plasmid into 3x 20 ul eppendorfs for front bench in ice bucket or cold block.
9. thaw whatever M13.1 plasmids are available to study and leave on front bench in ice bucket or cold block.
10. aliquot 3x 50 ul Kanamycin and leave on front bench in ice bucket or cold block.
11. aliquot 6x 10 ml LB into falcon tubes and leave on front bench.
12. bring full rack of sterile large tubes to front bench.

At end of lab:

1. move tube with LB+Kan to 4° until needed tomorrow.

Day after lab:

1. Remove plates from incubator and blow plug into LB+Kan. Grow on roller wheel 37° ON.

Notes: Day 5

Before lab:

Day of lab:

1. Need quiz.

Notes:Day 6

Before lab:

Day of lab:

Notes: Day 7

Oral Presentations

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°.

Inoue TB

1. Used for making Blattner strains competent.
2. 10 mM Pipes, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl.
3. All the components except the MnCl2 were mixed and the pH adjusted to 6.7 with KOH then MgCl2 was dissolved and the solution was filter sterilized.
4. For Fall of 2007 we have 1L in lab already.