20.109(F15): TA notes for module 2

From Course Wiki
Jump to: navigation, search

General notes

Key preparation:

  • Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
  • Need to streak out DE3 and DE3/IPC(wild-type) from frozen stocks in advance of liquid culture setup.
    • To be most careful, may want to freshly transform DE3 with pRSET-IPC miniprep.
    • DE3 in collection is NB301/AB2
    • DE3 w/IPC is NB303/AB4

Scheme: each pair of students will make one IPC mutant.

Daily notes

Day 1

Materials required:

  • Primers for mutagenesis, forward and reverse, premixed at 10 μM.</font color>
  • pRSET-IPC at 25 ng/μL (1 μL per group)
  • NEB 5α competent cells (one 50-μL aliquot per team)
  • Q5 master mix
  • Q5 KLD mix and buffer
  • SOC (1 mL per group)
  • Enzymes (NaeI or NgoMIV, and StyI for diagnostics may need to be ordered as well, if diagnostics digests performed on M2D2.

Preparation

  • PCR tubes on racks obtained from the freezer
  • Prepare a few aliquots of Master Mix for students, plus a control reaction

Primers

Mutation (X#Z) Forward primer (5' - 3') Reverse primer (5' - 3')
D21A ATT CGA CAA GGC TGG GGA CGG CA AAT GAG AAG GCT TCT TTG AAC TCT GC
E30K CAC CAC AAA GAA ACT TGG CAC CG ATG GTG CCG TCC CCA TCC
L31R CAC AAA GGA ACG TGG CAC CGT TAT G GTG ATG GTG CCG TCC CCA
D57H AGT CGA TGC TCA TGG CAA TGG AA TCA TTG ATC ATA TCC TGC AAT TC
T78P AAT GAA GGA CCC AGA CAG CGA AG TTT CTA GCC ATC ATA GTA AGA AAT TC
D93V CCG TGT TTT TGT CAA GGA TGG GAA C AAT GCT TCT CGG ATT TCC TC
M123L AGT TGA TGA ATT GAT AAG GGA AGC TCT TCA TCT GTT AAC TTC TCC
D130G AGC AGA TAT CGG TGG TGA TGG CC TCC CTT ATC ATT TCA TCA ACT TCT TCA TC
D132H TAT CGA TGG TCA TGG CCA AGT AAA C TCT GCT TCC CTT ATC ATT TC

At end of day: freeze SDM DNA

Preparation for M2D2

  • KLD digestion (by DpnI) of class mutants (1 h)
  • Transform NEB 5α cells, and plate on LB+Amp plates
  • Inoculate all class mutants
  • Mini-prep all mutants (2 colonies per group)
  • Streak BL21(DE3) cells on one LB+Cam plate
  • Finally, at M2D2 J-1, inoculate BL21(DE3) cells in LB+Cam

Day 2

Materials required:

  1. Bacterial transformation
    • calcium chloride: 1.47 g of CaCl2 dehydrate in 100 mL water, filter sterilize
    • LB+Amp+Cam plates - > 1.5 L of LB, separated into 2 flasks for 30 min of autoclave
    • cultures of competent BL21 cells at OD 0.4-0.6
      • if overnight culture has an OD600 of 1.5, then a 1:100 subculture reaches exponential growth phase (OD600 = 0.4-0.8) in 3 hours
  2. Sequencing reactions
    • nuclease-free water (30 μL per team)
    • mini-prep'ed DNA (each team will use 10 μL of each of 2 mutants + 2 μL of wt IPC)
    • IPC-F2 and IPC-R forward and reverse sequencing primers
      • Dilute each primer's 100 μM stock 1:5 in water (i.e. 80 μL water + 20 μL primer)
      • Thus primers at 5 pmol/μL.

Day of lab

  • Pre-warm water bath to 42 °C
  • Pre-warm LB medium
  • Pre-warm LB+Amp+Cam plates (4 per team)
  • Put CaCl2 on ice (6 mL per team)

Day 3

Ahead of lab:

  • For each team, inoculate wild-type IPC, X#Z #1 and X#Z #2 mutants.
  • 3 hours before lab, subculture these cells 1:20 in LB+Amp+Cam.
  • Aliquot freshly made IPTG (200 μL per team).
    • 0.1 M stock = 0.2383 g IPTG (stored at 4 °C) in 10 mL water

On instructors' bench:

  • cuvettes for spectrophotometer
  • LB for blank, as well as for 1:10 dilutions before reading OD600

On students' benches:

  • 3 cell samples in glass culture tubes
  • IPTG
  • ice bucket (to keep -IPTG samples cold throughout lab duration)
  • four plates of BL21 cells (transformed on M2D2 with wt IPC, mutant #1, mutant #2, or no DNA)

Day 4

Ahead of lab: Aliquot

  • EasyLyse bacterial protein extraction solution:
    • 25 mL of EasyLyse
    • 250 μL of 10% BSA, stored at -20 °C
    • 125 μL of protease inhibitor cocktail III (recommended at 1:200), stored at -20 °C
    • Aliquot 2 mL per team
  • Laemmli buffer: aliquot 20 μL per team
  • Protein purification buffers: aliquot per team
    • Resin: 900 μL
    • Charge buffer: 2.5 mL (mix 4 mL of 8x stock with 28 mL water)
    • Binding buffer: 5.5 mL (mix 8.25 mL of 8X stock with 57.75 mL water)
    • Wash buffer: 2.5 mL (mix 4 mL of 8x stock with 28 mL water)
    • Elute buffer: 2.5 mL (mix 7.5 mL of 4x stock with 22.5 mL water)
  • Bradford assay: aliquot per team
    • 12 mL water
    • 2.5 mL Bradford reagent
    • BSA standards:
      • Make 1:100 of 10% BSA stock for 0.1% BSA dilution stock by mixing 693 μL water + 7 μL 10% BSA
      • BSA 0.1mg/mL: 10 μL (0.1% BSA, 20 μL in 180 μL water = 200 μL)
      • BSA 0.2mg/mL: 10 μL (0.1% BSA, 40 μL in 160 μL water = 200 μL total)
      • BSA 0.4mg/mL: 10 μL (0.1% BSA, 80 μL in 120 μL water = 200 μL total)
      • BSA 0.6mg/mL: 10 μL (0.1% BSA, 120 μL in 80 μL water = 200 μL total)
      • BSA 0.8mg/mL: 10 μL (0.1% BSA, 160 μL in 40 μL water = 200 μL total)
      • BSA 1.0mg/mL: 10 μL (0.1% BSA, 200 μL)

On instructors' bench:

  • lysing enzyme
  • filtered tips for P20
  • extra 15 mL conical tubes
  • nutator
  • cuvettes for spectrophotometer
  • Laemmli buffer
  • bucket to collect waste tubes (Charge-Nickel, Imidazole, and Bradford-methanol)

On students' benches: On ice:

  • all aliquots (see "Ahead of lab above")
  • 6 pellets: -/+ IPTG of wt IPC, mutant #1 and mutant #2
  • 2 Zeba desalting columns
  • Charge Buffer waste 15 mL tube
  • imidazole waste 15 mL tube
  • Bradford waste 15 mL tube

Day 5

Electronics:

  • charged computer with student PowerPoint presentations on desktop
  • adapter to projector if needed
  • video-camera to record students
  • laser pointer
  • timer(s)
  • grading charts with rubric

Treats:

  • plates, cups, forks and napkins
  • juice and tea
  • snacks

Day 6

On instructors' bench:

  • Lid locks
  • Students' samples to be boiled
  • Students' purified proteins to be titrated
  • Black 96-well plates, 1 per team
  • Multichannel pipets
  • Filtered tips (P200)
  • 1 mL water mixed with 10 μL of 10% BSA, per team
  • 12 calcium reservoirs
    • Instructor teaches how to do the multichannel pipetting (avoid bubbles, make sure all tips are actually taking up the same volume, etc).
    • 2 mL of calcium solution in each reservoir (150 μL / solution / team)
reservoir # final [Ca2+]ifree (nM) volume of zero buffer (μL) volume of high calcium buffer (μL)
1 0 1000 0
2 8.5 900 100
3 10 800 200
4 32.5 700 300
5 50 600 400
6 75 500 500
7 112.5 400 600
8 175.5 300 700
9 301 200 800
10 675 100 900
11 1505 50 950
12 19500 0 1000

In hood:

  • Water baths with boiling chips, turn early on in lab
  • Waste bottle for Coomassie stain

On gel bench:

  • Polyacrylamide gels (1 per pair).
  • TGS buffer (1 L per box)
  • Staining boxes
  • Coomassie bottle and 50 mL conical tubes for measuring
  • Distilled water

After lab:

  • Post data to wiki.