20.109(F11): TA's notes for module 3

Well in advance of Day 1

Make up overnight inoculum of phage for Thursday class: 20 mL LB, 20 µL XL1-blue (can also grow on strain "4-1" (ER2738) to slightly higher titer, 20 µL tet, 20 µL virus (from supernatant or PEG precipitated); divide between 4 tubes rotate at 37ºC overnight. Store
autoclave/sterilize 16 125-mL flasks (one flask per group)
make sure there are enough Oak Ridge tubes for each group (at least 2 per group)
make sure that there is a centrifuge available for Tues and Wed afternoon for the PEG precipitation (Niles lab down the hall)
make enough PEG solution (20% PEG-8000, 2.5 M NaCl --- this requires a long time to fully solubilize - leave on the stir plate for about an hour). Each group will need <10 ml.
make 10 mM NaCl, pH 5.3 (need 2L/lab)
make 10 mM NaCl, pH 10 (need 1L/lab)

TUESDAY LAB: start Saturday before Day 1

Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks (for Tuesday class). Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Monday night). Let stand on bench overnight until class on Tuesday.

WEDNESDAY LAB: start Monday before Day 1

Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks (for Tuesday class). Add 1 mL of overnight inoculum (can use Tuesday lab's larger cultures if needed), and incubate shaking at room temperature for two days (til Wednesday class). Let stand on student bench or front bench at start of class.

no quiz since first day of module
Check-in (again) with Niles lab about using centrifuge
ice buckets (three) filled with ice for PEG precipitation, one on each student bay.
40 mL phage cultures, one per pair of students.
Oak Ridge Tubes, 2/pair of students.
20 % PEG/ 2.5M NaCl, about 10 ml/pair of students. Keep in an ice bucket on front bench.
1X TBS (USE ROCKLAND not BioRAD!), each pair of students will need less than 5 ml. Aliquot into 15 ml falcon tubes and leave on front bench.
Aliquots of water, each pair of students will need less than 5 ml. Aliquot into 15 ml falcon tubes and leave on front bench.
Set out quartz cuvettes on front teacher's bench. Turn on UV lamps on spec about 1/2 way through lab.
set out dialysis tubes, clips, falcon tubes for soaking tubing (students can get H2O as needed), and SWNTs from Belcher lab
at end of lab set up 10 mM NaCl, pH 5.3 in beaker with stir bar on stir plate. Tape dialysis bags to edge of beaker if needed.

AFTER LAB

Need to replace pH 5.3 NaCl with fresh 5.3 after about 2 hrs and dialyze overnight.
NEXT DAY: replace pH 5.3 NaCl with pH 10 and dialyze overnight.

need quiz
make sure hood is clean and place 3x stir plates in hood
set out 3x 15 ml falcon tubes (one per "super group") and 3x 50 ml falcon tubes, on teacher's bench
set out top loading balance on teacher's bench, and bucket or two of ice, plus some large weigh boats
set out evaporation dishes (3), and small evaporation jars with lids (3) on teacher's bench
chill 50 ml of 100% EtOH on ice
midway through lab set out TEM grids and tweezers (enough for every pair of students), as well as 3x 1 ml aliquots of 100% EtOH and 3x 1ml aliquots of sterile water
set out 100 ml bottles of sterile water for washing nanowires