20.109(S16): TA notes for module 1

From Course Wiki
Revision as of 12:49, 12 February 2016 by MAXINE JONAS (Talk | contribs)

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: 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

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.
Retrieved from "http://measurebiology.org/w/index.php?title=20.109(S16):_TA_notes_for_module_1&oldid=30007"