Difference between revisions of "20.109(S16): TA notes for module 1"

Latest revision as of 17:12, 2 March 2016

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)

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