20.109(S07): Biomaterial engineering, TA notes

Module 4 Day 1

1. no quiz
2. Student Benchtop:
   • • Three cultures, 5mL of each, for each group
     • Overnight of NY290 (pCT-CON in “DY”)
     • Overnight of NY291 (pAu1 in “DY”)
     • Overnight of NY286 (library screen)
     • 50mL Gal blocking/binding buffer
     • 3 falcon tubes
     • 2 six-well dishes
     • Pipet bulbs
     • Forcepts
     • Screw cap jar 100% EtOH
     • Kimwipes
3. Instructor Bechtop:
   • • Extra cultures
     • Extra blocking buffer
     • Sleeve of 10 and 5mL pipets
     • -trp plates (3/group = 18 at least)
     • Gold slides cut into 6mm x 10mm (3 pieces/group = 18 pieces at least)
     • Digital camera, batteries charged

INSTRUCTIONS

1. At least a week before the class begins: streak out the following strains
   • • NY290 on –trp
     • NY291 on –trp
2. Two days before lab: set up the following as 2x 2.5mL SC-trp at 30∞
   • • NY290= pCT-CON in “DY” = EBY100
     • NY291= pAu1 in “DY” = EBY100
3. The day before lab: set up 50mL in RT shaker, inoculating 1.25mL into each
   • • NY290 in CAA(Gal)
     • NY291 in CAA (Gal)
4. To grow the library go directly from the frozen stock of NY286 and inoculate 5mL of CAA(Gal) for each group, plus one or two extra in case someone drops a tube. Use a good size inoculum.

Module 4 Day 2

1. Need quiz
2. Student Benchtop:
   • • Two cultures, 2 x 5mL of each, for each group
     • Overnight of NY290 (pCT-CON in “DY”)
     • Overnight of NY291 (pAu1 in “DY”)
     • 50mL Gal blocking/binding buffer
     • 4 falcon tubes
     • 3 six-well dishes
     • Pipet bulbs
     • Forcepts
     • Screw cap jar 100% EtOH
     • Kimwipes
3. Instructor Bechtop:
   • • Extra cultures
     • Extra blocking buffer
     • Sleeve of 10 and 5mL pipets
     • -trp plates (6/group = 36 at least)
     • Gold slides cut into 6mm x 10mm (6 pieces/group = 36 pieces at least)
     • Rack of sterile 16mm tubes for students to set up overnights
     • Sterile SC(Glu)-trp for students to inoculate cultures
     • Sterile CAA(Gal) for students to aliquot
     • Digital camera, batteries charged

INSTRUCTIONS

1. Grow NY290 and NY291 cultures in CAA(Gal) precisely as done for Module 4 Day 1 except set up twice as many for each lab.

Module 4 Day 3

1. Need quiz
2. Student Benchtop:
   • • Four cultures, 5mL of each, induced from library screen
     • Two cultures, 5mL of each, for each group
     • Overnight of NY290 (pCT-CON in “DY”)
     • Overnight of NY291 (pAu1 in “DY”)
     • 50mL Gal blocking/binding buffer
     • 6 falcon tubes
     • 3 six-well dishes
     • Pipet bulbs
     • Forcepts
     • Screw cap jar 100% EtOH
     • Kimwipes
3. Instructor Bechtop:
   • • Extra cultures
     • Extra blocking buffer
     • Sleeve of 10 and 5mL pipets
     • -trp plates (6/group = 36 at least)
     • Gold slides cut into 6mm x 10mm (6 pieces/group = 36 pieces at least)
     • Digital camera, batteries charged

INSTRUCTIONS

1. One day before lab: induce each student culture by moving 125uL into the corresponding 5mL of CAA(Gal). Also induce 50mL of CAA(Gal) with 1.25mL controls. Grow RT O/N.

Module 4 Day 4

1. Need quiz
2. Student Benchtop:
   • • Ice buckets with ice
     • Toothpicks
     • Sterile water
3. Instructor Bechtop:
   • • Ice bucket with PCR master mix, PCR primers (called NO52 and NO53), positive control DNA
     • For sequencing, you want each tube to have 2uL of 1:10 dilution of PCR product, 6.4uL of 1:100 seq primer (called NO50) and 15.6uL water. Can mix the last two as a cocktail and aliquot 22uL / seq tube.

INSTRUCTIONS

1. This lab time will be straightforward, with just PCR, and then running samples to the biopolymer facility (E17-415) and thankfully there are no cultures to prepare in advance.
2. PCR program “called something like 52/35”
   • 94∞4’
   • 94∞1’
   • 52∞1
   • 72∞2’ 35x
   • 72∞10’
   • 4∞ hold
3. DNA seq request form is at

http://web.mit.edu/biopolymers/www/request_form.html

Module 4 Day 5

Student presentations

Module 4 Day 6

1. Need quiz
2. The set-up for this lab is nothing. Just confirm that the laptops are all working and have the appropriate programs to retrieve sequence data from the Biopolymer’s facility.
3. For the PCs, this would mean FileZila to retrieve sequences
4. Chromas http://www.technelysium.com.au/chromas.html to view traces
5. Excel to view sequence
6. IE or Netscape to analyze it

RECIPES

• YPD
  • 20g Peptone
  • 10g Yeast Extract
  • 20g agar, if making plates
  • add 950mL distilled H2O and stir bar
  • autoclave 30’ at most
  • add 50mL 40% glucose when cool-ish

• Drop out plates from Q-BIOGENE mixes

• CAA (GAL)
  • 10g Galactose
  • 3.35g YNB w/o aa (Difco 291940)
  • 2.7g Na2HPO4~7H2O (Sigma S9390)
  • 4.28g NaH2PO4~H2O (Sigma S9638)
  • 5g CAA (BactoDifco 2230050)
  • 500mL distilled H2O
  • filter sterilize

• GAL Binding/Blocking Buffer – make the day of lab
  • To 500mL of CAA (GAL) add 2.5g BSA and 5mL 10% Tween. Dissolve by inverting several times. Aliquot 50mL/student group.

• SC-trp as directed from BIO-101