20.109(S16): TA notes for module 1
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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:
- 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
- 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)
- 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 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.