20.109(F15): TA notes for module 2
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Contents
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
Mutation (X#Z) | Reverse primer (5' - 3') |
D21A | AAT GAG AAG GCT TCT TTG AAC TCT GC |
E30K | ATG GTG CCG TCC CCA TCC |
L31R | GTG ATG GTG CCG TCC CCA |
D57H | TCA TTG ATC ATA TCC TGC AAT TC |
T78P | TTT CTA GCC ATC ATA GTA AGA AAT TC |
D93V | AAT GCT TCT CGG ATT TCC TC |
M123L | TCT TCA TCT GTT AAC TTC TCC |
D130G | TCC CTT ATC ATT TCA TCA ACT TCT TCA TC |
D132H | TCT GCT TCC CTT ATC ATT TC |
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:
- 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
- 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
- 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 |
On students' benches:
- Multichannel pipet
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
Materials required:
- 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.