20.109(S07): Biomaterial engineering, TA notes

Module 4 Day 1

 * 1) no quiz
 * 2) Student Benchtop:
 * 3) **Three cultures, 5mL of each, for each group
 * 4) **Overnight of NY290 (pCT-CON in “DY”)
 * 5) **Overnight of NY291 (pAu1 in “DY”)
 * 6) **Overnight of NY286 (library screen)
 * 7) **50mL Gal blocking/binding buffer
 * 8) **3 falcon tubes
 * 9) **2 six-well dishes
 * 10) **Pipet bulbs
 * 11) **Forcepts
 * 12) **Screw cap jar 100% EtOH
 * 13) **Kimwipes
 * 14) Instructor Bechtop:
 * 15) **Extra cultures
 * 16) **Extra blocking buffer
 * 17) **Sleeve of 10 and 5mL pipets
 * 18) **-trp plates (3/group = 18 at least)
 * 19) **Gold slides cut into 6mm x 10mm (3 pieces/group = 18 pieces at least)
 * 20) **Digital camera, batteries charged

INSTRUCTIONS

 * 1) At least a week before the class begins: streak out the following strains
 * 2) **NY290 on –trp
 * 3) **NY291 on –trp
 * 4) Two days before lab: set up the following as 2x 2.5mL SC-trp at 30∞
 * 5) **NY290= pCT-CON in “DY” = EBY100
 * 6) **NY291= pAu1 in “DY” = EBY100
 * 7) The day before lab: set up 50mL in RT shaker, inoculating 1.25mL into each
 * 8) **NY290 in CAA(Gal)
 * 9) **NY291 in CAA (Gal)
 * 10) 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:
 * 3) **Two cultures, 2 x 5mL of each, for each group
 * 4) **Overnight of NY290 (pCT-CON in “DY”)
 * 5) **Overnight of NY291 (pAu1 in “DY”)
 * 6) **50mL Gal blocking/binding buffer
 * 7) **4 falcon tubes
 * 8) **3 six-well dishes
 * 9) **Pipet bulbs
 * 10) **Forcepts
 * 11) **Screw cap jar 100% EtOH
 * 12) **Kimwipes
 * 13) Instructor Bechtop:
 * 14) **Extra cultures
 * 15) **Extra blocking buffer
 * 16) **Sleeve of 10 and 5mL pipets
 * 17) **-trp plates (6/group = 36 at least)
 * 18) **Gold slides cut into 6mm x 10mm (6 pieces/group = 36 pieces at least)
 * 19) **Rack of sterile 16mm tubes for students to set up overnights
 * 20) **Sterile SC(Glu)-trp for students to inoculate cultures
 * 21) **Sterile CAA(Gal) for students to aliquot
 * 22) **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:
 * 3) **Four cultures, 5mL of each, induced from library screen
 * 4) **Two cultures, 5mL of each, for each group
 * 5) **Overnight of NY290 (pCT-CON in “DY”)
 * 6) **Overnight of NY291 (pAu1 in “DY”)
 * 7) **50mL Gal blocking/binding buffer
 * 8) **6 falcon tubes
 * 9) **3 six-well dishes
 * 10) **Pipet bulbs
 * 11) **Forcepts
 * 12) **Screw cap jar 100% EtOH
 * 13) **Kimwipes
 * 14) Instructor Bechtop:
 * 15) **Extra cultures
 * 16) **Extra blocking buffer
 * 17) **Sleeve of 10 and 5mL pipets
 * 18) **-trp plates (6/group = 36 at least)
 * 19) **Gold slides cut into 6mm x 10mm (6 pieces/group = 36 pieces at least)
 * 20) **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:
 * 3) **Ice buckets with ice
 * 4) **Toothpicks
 * 5) **Sterile water
 * 6) Instructor Bechtop:
 * 7) **Ice bucket with PCR master mix, PCR primers (called NO52 and NO53), positive control DNA
 * 8) **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
http://web.mit.edu/biopolymers/www/request_form.html
 * 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”
 * 3) *94∞4’
 * 4) *94∞1’
 * 5) *52∞1
 * 6) *72∞2’ 35x
 * 7) *72∞10’
 * 8) *4∞ hold
 * 9) DNA seq request form is at

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