Difference between revisions of "20.109(F15): TA notes for module 2"

Latest revision as of 12:11, 3 November 2015

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

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)

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.