20.109(F07): TA's notes for module 3: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 8: | Line 8: | ||
# 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 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 Blattner minimal strains on LB plate at 37° ON. These are NB258 through NB263. | # Wake up Blattner minimal strains on LB plate at 37° ON. These are NB258 through NB263. | ||
# Wake up M13 | # Wake up M13 titering 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'. | ||
# Pour LB plates (4L), 4 | # Pour LB plates (4L), 6 plates per group, x 2 days. | ||
# Prepare top agar if needed. | # Pour LB+ Kan plates (2L), 4 plates per group, x 2 days. | ||
# Prepare top agar if needed. TA left over from Mod 1 will still work as long as it's not contaminated. | |||
# Autoclave more large and small tubes if necessary. | # Autoclave more large and small tubes if necessary. | ||
# Check stock of LB liquid media and make more if necessary. | # Check stock of LB liquid media and make more if necessary. | ||
Line 20: | Line 21: | ||
# Grow titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group. | # Grow titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group. | ||
Day of lab: | Day of lab: | ||
# | # Do no need quiz on day 1 but in future all quizzes 2 or 3 questions, 5 points. | ||
# Aliquot PEG solution for each group. | # Aliquot PEG solution for each group. (500 ul/group in eppendorfs should be enough) | ||
# Place rack of 15 ml falcon tubes and foil out for Ir pH'ing. | # 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 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 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 | # 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: | Day after lab: | ||
# Remove petri dishes from 37° incubator and store at 4° or RT until next time. | # Remove petri dishes from 37° incubator and store at 4° or RT until next time. | ||
Line 35: | Line 37: | ||
Day of lab: | Day of lab: | ||
# Need quiz | # Need quiz | ||
# | # Distribute titer plates from Day 1 | ||
# Place electropulser on bench with balance. | |||
# Place cuvettes where students can collect as needed. | |||
# Place nutator on bench across from balance with foil to cover. | |||
# Place dialysis tubing and clamps on teacher demo area. | |||
===[[20.109(F07): Transmission electron microscopy | Notes: Day 3]]=== | ===[[20.109(F07): Transmission electron microscopy | Notes: Day 3]]=== |
Revision as of 13:40, 12 October 2007
Biomaterials Engineering Module
no wiki archive version
General notes
Before term/module begins:
- 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.
- 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.
- Confirm pH meter is in good working order.
- 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 Blattner minimal strains on LB plate at 37° ON. These are NB258 through NB263.
- Wake up M13 titering 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'.
- Pour LB plates (4L), 6 plates per group, x 2 days.
- Pour LB+ Kan plates (2L), 4 plates per group, x 2 days.
- Prepare top agar if needed. TA left over from Mod 1 will still work as long as it's not contaminated.
- Autoclave more large and small tubes if necessary.
- Check stock of LB liquid media and make more if necessary.
- 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:
- 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 titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group.
Day of lab:
- Do no 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)
- 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.
Notes: Day 2
Before lab:
Day of lab:
- Need quiz
- Distribute titer plates from Day 1
- Place electropulser on bench with balance.
- Place cuvettes where students can collect as needed.
- Place nutator on bench across from balance with foil to cover.
- Place dialysis tubing and clamps on teacher demo area.
Notes: Day 3
Before lab:
Day of lab:
- no quiz since students come to lab at staggered times to get to TEM at different times.
Notes: Day 4
Before lab:
Day of lab:
- Need quiz.
Notes: Day 5
Before lab:
Day of lab:
- Need quiz.
Notes:Day 6
Before lab:
Day of lab:
Notes:Day 7
Before lab:
Day of lab:
Notes: Day 8
Recipes/Reagents
- 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°.
- 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.
- 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°.