20.109(S16): TA notes for module 1

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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: In ice bucket:

  • pRSET-IPC at 500 ng/μL (20 μL per group)
  • restriction enzymes
  • NEB buffers
  • water

Day 2

In the morning before lab: Pour agarose gels:

  • 100 mL TAE + 1 g agarose + 10 uL SYBR-Safe
  • 14-tooth combs
  • 4 teams per gel

On instructors' bench:

  • In ice bucket:
    • students' confirmation digests (4 per team: uncut, enzyme 1, enzyme 2, double digest)
    • loading dye: aliquot 25 uL per team
    • 1 kb ladder

Day 3

On instructors' bench:

  • primers for mutagenesis, forward and reverse, in powder form
  • nuclease-free water, aliquots of 1.5 mL per team
  • pRSET-IPC at 25 ng/μL (1 μL per group)
  • Q5 master mix
  • ... and for the end of the day, for instructors:
    • NEB 5α competent cells (one 50-μL aliquot per team)
    • Q5 KLD mix and buffer
    • warm SOC (1 mL per group)
    • LB+Amp plates (1 per group + 1 control)

Preparation

  • PCR tubes on racks obtained from the freezer
  • filtered tips
  • control SDM reaction

Preparation for M1D3

  • KLD digestion (by DpnI) of class mutants (1 h)
  • Transform NEB 5α cells, and plate on LB+Amp plates
  • Inoculate all class mutants

Ahead of Day 4

  • D4-2:
    • possibly repeat students' (failed) SDM reactions
    • streak BL21(DE3)pLysS cells on LB+Cam plate
  • D4-1
    • inoculate 2 colonies of mutant pRSET-IPC in NEB 5α cells for each team (in LB+Amp broth)
    • inoculate BL21 cells (in LB+Cam broth)

Day 4

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 (5 mL per team) 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. Mini-prep
    • aliquot Qiagen kit: P1 (600 μL per team), P2 (600 μL), N3 (750 μL), PB (1.1 mL), PE (1.6 mL), water pH 8.0 (75 μL)
  3. 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 1:20 in water (i.e. 95 μL water + 5 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)
  • Set out glass culture tubes (3 per team)

Day 5

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 6

Ahead of lab: Aliquot

  • BugBuster bacterial protein extraction solution:
    • 30 mL of BugBuster
    • 300 μL of 10% BSA, stored at -20 °C
    • 150 μL of protease inhibitor cocktail III (recommended at 1:200), stored at -20 °C
    • Aliquot 2 mL per team
  • Laemmli buffer: aliquot 40 μL per team
  • Protein purification buffers: aliquot per team
    • Resin: 900 μL
    • Binding buffer: 3.5 mL (mix 10 mL of 5X stock with 40 mL water)
    • Wash buffer: 4.5 mL (mix 13 mL of 5x stock with 52 mL water)
    • Elute buffer: 2.5 mL (mix 8 mL of 5x stock with 32 mL water)
  • Micro BCA assay: aliquot per team
    • 25 mL water
    • 250 μL of 2.0 mg/mL BSA (brown ampoules in kit box)
    • 5.5 mL MA
    • 5.75 mL MB
    • 300 μL MC

On instructors' bench:

  • lysing enzyme (on ice)
  • 2 mL eppendorf tubes (to wash nickel beads, and for Micro BCA)
  • 5 mL "flow cytometry" tubes (Micro BCA)
  • filtered tips for P20
  • extra 15 mL conical tubes
  • nutator (also one nutator at 4 °C)
  • cuvettes for spectrophotometer
  • Laemmli buffer
  • bucket to collect waste tubes (Imidazole, and Micro BCA)
  • large water bath at 60 °C

On students' benches: On ice:

  • all aliquots (see "Ahead of lab above")
  • 4 pellets: -/+ IPTG of wt IPC, and mutant
  • 2 Zeba desalting columns
  • imidazole waste 15 mL tube
  • Micro BCA waste 50 mL tube

Day 7

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.

Day 8

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