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

Revision as of 20:38, 22 October 2015

Long overdue for a revision

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

Reverse primers

At end of day: freeze SDM DNA

How it went:

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 uL of 10% BSA, stored at -20 °C
  • 125 uL of protease inhibitor cocktail III (recommended at 1:200), stored at -20 °C
  • Aliquot 2.1 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
• bucket to collect waste tubes (Charge-Nickel, Imidazole, and Bradford-methanol)

On students' benches:

Day 6

Materials required:

Stuff requiring advance thought/prep: protein purification buffers, BSA standards

• Part 1: cell lysis, all up at teaching bench
  • BPER (3 mL aliquots, 30 μL 10% BSA and 30 μL protease inhibitors)
    • last-minute prep, as it should be kept at room temp but has enzymes in it
  • Lysis enzyme available on ice up front
• Part 2: SDS-PAGE advance prep
  • Water (150 μL aliquots)
  • 2X sample buffer available in hood
    • about 100μL group, three 300 μL to share should be fine
    • add 5% β-Me at last minute
• Part 3A: protein purification (see also googledoc!), in ice buckets at their benches
• Nu
  • Note: prepare solutions ~15% in excess of needed volume
  • Water, Charge Buffer
  • Binding Buffer, Wash Buffer, and Elution Buffer with protease inhibitors
  • Small BSA aliquots ready.
• Part 3B: protein purification
• Part 4: protein concentration, at teaching bench
  • put out closer to end of lab
  • 5X Coomassie stain from Bio-Rad out, three 8 mL aliquots to share (each team needs 2.4 mL)
  • 9.6 mL aliquot of water, one per team (plus an extra)
  • BSA standards --

Day of Lab:

• Quiz

MOVING ELSEWHERE

• Transfer gels to fresh water at end of lab and/or next day.
• Collect all purified protein samples from students and store at 4 °C.

Day after Lab:

• Transfer gels to water and take pictures.
• Put up sign in BPEC reserving Day 7 platereader use.

How it went:

• This is another long day that may need a bit more black-boxing/efficiency work.

Day 7

Materials required:

1. Pipetting reservoirs - 2 per group
2. Calcium solutions - 0.5 mL/soln/group

Day of Lab:

1. SDS-PAGE, gel bench
   • Polyacrylamide gels (1 per pair).
   • TGS buffer (1 L per box)
   • Staining boxes, couple of spatulas
   • Coomassie bottle and 50 mL conical tubes for measuring
   • Distilled water in 1 L bottles
2. SDS-PAGE, in hood
   • Water baths with boiling chips, turn early on in lab
   • Lid locks
   • Waste bottle for stain

• Quiz (prepared by TA).
• Post data to wiki.
• Instructor teaches the first group of students how to do the multichannel pipetting (avoid bubbles, make sure all tips are actually taking up the same volume, etc.); then TA takes over while instructor is in BPEC for plate reading.

How it went:

• Protein amounts (fluorescence values) were pretty consistently higher this year than in year's past. In part may be due to using Invitrogen instead of Novagen resin, due to a mix-up; in part may be due to the higher cell ODs for many students. Perhaps in future should aim for high-log rather than mid-log growth, in fact.

Day 8

Materials required:

1. None: all computer work today.

Day of Lab:

• Quiz (prepared by TA).

How it went:

• M124S a much better parallel sample than S101L (run in S09). The change in affinity and cooperativity was dramatic and consistent across the class.
• The only strange issue is that at very low calcium concentrations the data is noisy rather than simply being a high plateau, or even somewhat consistently starts a little low, then has the high plateau, then proceeds as expected.