Difference between revisions of "20.109(F15): TA notes for module 2"
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==General notes== | ==General notes== | ||
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*Prepare a few aliquots of Master Mix for students, plus a control reaction | *Prepare a few aliquots of Master Mix for students, plus a control reaction | ||
− | ''' | + | '''Primers''' |
− | + | <center> | |
{| border="1" | {| border="1" | ||
|'''Mutation (X#Z)''' | |'''Mutation (X#Z)''' | ||
+ | |'''Forward primer (5' - 3')''' | ||
|'''Reverse primer (5' - 3')''' | |'''Reverse primer (5' - 3')''' | ||
|- | |- | ||
| D21A | | D21A | ||
+ | | ATT CGA CAA GGC TGG GGA CGG CA | ||
| AAT GAG AAG GCT TCT TTG AAC TCT GC | | AAT GAG AAG GCT TCT TTG AAC TCT GC | ||
|- | |- | ||
| E30K | | E30K | ||
+ | | CAC CAC AAA GAA ACT TGG CAC CG | ||
| ATG GTG CCG TCC CCA TCC | | ATG GTG CCG TCC CCA TCC | ||
|- | |- | ||
| L31R | | L31R | ||
+ | | CAC AAA GGA ACG TGG CAC CGT TAT G | ||
| GTG ATG GTG CCG TCC CCA | | GTG ATG GTG CCG TCC CCA | ||
|- | |- | ||
| D57H | | D57H | ||
+ | | AGT CGA TGC TCA TGG CAA TGG AA | ||
| TCA TTG ATC ATA TCC TGC AAT TC | | TCA TTG ATC ATA TCC TGC AAT TC | ||
|- | |- | ||
| T78P | | T78P | ||
+ | | AAT GAA GGA CCC AGA CAG CGA AG | ||
| TTT CTA GCC ATC ATA GTA AGA AAT TC | | TTT CTA GCC ATC ATA GTA AGA AAT TC | ||
|- | |- | ||
| D93V | | D93V | ||
+ | | CCG TGT TTT TGT CAA GGA TGG GAA C | ||
| AAT GCT TCT CGG ATT TCC TC | | AAT GCT TCT CGG ATT TCC TC | ||
|- | |- | ||
| M123L | | M123L | ||
+ | | AGT TGA TGA ATT GAT AAG GGA AGC | ||
| TCT TCA TCT GTT AAC TTC TCC | | TCT TCA TCT GTT AAC TTC TCC | ||
|- | |- | ||
| D130G | | D130G | ||
+ | | AGC AGA TAT CGG TGG TGA TGG CC | ||
| TCC CTT ATC ATT TCA TCA ACT TCT TCA TC | | TCC CTT ATC ATT TCA TCA ACT TCT TCA TC | ||
|- | |- | ||
| D132H | | D132H | ||
+ | | TAT CGA TGG TCA TGG CCA AGT AAA C | ||
| TCT GCT TCC CTT ATC ATT TC | | TCT GCT TCC CTT ATC ATT TC | ||
|- | |- | ||
|} | |} | ||
− | + | </center> | |
At end of day: freeze SDM DNA | At end of day: freeze SDM DNA | ||
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'''Preparation for M2D2''' | '''Preparation for M2D2''' | ||
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*6 pellets: -/+ IPTG of wt IPC, mutant #1 and mutant #2 | *6 pellets: -/+ IPTG of wt IPC, mutant #1 and mutant #2 | ||
*2 Zeba desalting columns | *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 | ||
− | + | ===[[20.109(F15):Characterize protein expression (Day6)| 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) |
− | * | + | <center> |
− | + | {|border=1px | |
− | + | |'''reservoir #''' | |
− | + | |'''final [Ca<sup>2+</sup>]<sub>i</sub>free (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 | ||
+ | |} | ||
+ | </center> | ||
− | ''' | + | '''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:''' | |
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*Post data to wiki. | *Post data to wiki. | ||
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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
Mutation (X#Z) | Forward primer (5' - 3') | Reverse primer (5' - 3') |
D21A | ATT CGA CAA GGC TGG GGA CGG CA | AAT GAG AAG GCT TCT TTG AAC TCT GC |
E30K | CAC CAC AAA GAA ACT TGG CAC CG | ATG GTG CCG TCC CCA TCC |
L31R | CAC AAA GGA ACG TGG CAC CGT TAT G | GTG ATG GTG CCG TCC CCA |
D57H | AGT CGA TGC TCA TGG CAA TGG AA | TCA TTG ATC ATA TCC TGC AAT TC |
T78P | AAT GAA GGA CCC AGA CAG CGA AG | TTT CTA GCC ATC ATA GTA AGA AAT TC |
D93V | CCG TGT TTT TGT CAA GGA TGG GAA C | AAT GCT TCT CGG ATT TCC TC |
M123L | AGT TGA TGA ATT GAT AAG GGA AGC | TCT TCA TCT GTT AAC TTC TCC |
D130G | AGC AGA TAT CGG TGG TGA TGG CC | TCC CTT ATC ATT TCA TCA ACT TCT TCA TC |
D132H | TAT CGA TGG TCA TGG CCA AGT AAA C | TCT GCT TCC CTT ATC ATT TC |
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:
- 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
- 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.