20.109(F07): TA's notes for module 3: Difference between revisions
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==Biomaterials Engineering Module== | ==Biomaterials Engineering Module== | ||
[[20.109(F08):Module 3| 20.109(F08)]] <br> | |||
[[20.109(F07):Module 3| 20.109(F07)]] (archive) | |||
==General notes== | ==General notes== | ||
Before | Before module begins: | ||
# Check that all links on all wiki pages for biomaterial engineering | # 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. | ||
# | # Reserve TEM for Day 5 | ||
# 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 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'. | # 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'. | ||
# Pour LB plates | # Pour LB plates | ||
# Pour LB+ Kan plates | # Pour LB+ Kan plates | ||
# Prepare top agar if needed | # Prepare top agar if needed. | ||
# Autoclave more large and small tubes if necessary. | # Autoclave more large and small glass 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. | ||
===[[20.109( | ===[[20.109(F08): Mod 3 Day 1 Growth of phage materials| Day 1]]=== | ||
Before lab: | Before lab: | ||
# Grow phage infected strain NB273, enough for each pair of students to work with at least 3 ml. | # 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. | # 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. | # Grow NB271= titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group. | ||
Day of lab: | Day of lab: | ||
# Do | # Do not 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) | # Aliquot PEG solution for each group. (500 ul/group in eppendorfs should be enough) | ||
# Fill ice buckets for each group | |||
# 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. | # 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. | # 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. | ||
===[[20.109( | ===[[20.109(F08): Mod 3 Day 2 Phage nanowires| Day 2]]=== | ||
Before lab: | Before lab: | ||
Day of lab: | Day of lab: | ||
# Need quiz | # Need quiz | ||
# Distribute titer plates from Day 1 | # Distribute titer plates from Day 1 | ||
===[[20.109( | |||
Day after lab: | |||
===[[20.109(F08): Mod 3 Day 3 Journal Club II| Day 3]]=== | |||
*no prep since today is Journal Club (yay!) | |||
===[[20.109(F08): Mod 3 Day 4 Phage by design| Day 4]]=== | |||
Before lab: | Before lab: | ||
Day of lab: | Day of lab: | ||
*need quiz | |||
# | |||
At end of lab: | |||
Day after lab: | |||
===[[20.109(F08): Mod 3 Day 5 Phage by design, pt2| Day 5]]=== | |||
Day before lab: | |||
#Confirm reservation for TEM | |||
Day of lab: | Day of lab: | ||
# Need quiz. | # Need quiz. | ||
Day after lab: | |||
Day | |||
# | # | ||
===[[20.109( | ===[[20.109(F08): Mod 3 Day 6 ECD assembly| Day 6]]=== | ||
Before lab: | Before lab: | ||
# | # | ||
Line 71: | Line 76: | ||
===[[20.109( | ===[[20.109(F08): Mod 3 Day 7 Oral presentations| Day 7]]=== | ||
*no lab prep since this day is for presentations of research ideas (yay!) | |||
==Recipes/Reagents== | ==Recipes/Reagents== | ||
Line 79: | Line 85: | ||
# 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. | # 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. | ||
# Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates. | # Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates. | ||
# 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°. | # 20% PEG-8000/2.5M NaCl (salt dissolved in PEG solution) Store at 4°. | ||
Latest revision as of 03:06, 11 October 2008
Biomaterials Engineering Module
20.109(F08)
20.109(F07) (archive)
General notes
Before module begins:
- 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.
- Reserve TEM for Day 5
- 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 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'.
- Pour LB plates
- Pour LB+ Kan plates
- Prepare top agar if needed.
- Autoclave more large and small glass tubes if necessary.
- Check stock of LB liquid media and make more if necessary.
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 NB271= titering strain for M13 in LB+Kan. Grow at least 2.5 ml/lab group.
Day of lab:
- Do not 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)
- Fill ice buckets for each group
- 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.
Day 2
Before lab:
Day of lab:
- Need quiz
- 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:
- Confirm reservation for TEM
Day of lab:
- 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
- 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.
- 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.
- Kan: 25 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates.
- 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°.