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
- LB+Amp+Cam plates - > 1.5 L of LB, separated into 2 flasks for 30 min of autoclave
- autoclaved glass tubes
- cultures of competent BL21 cells at OD 0.4-0.6
Day of lab
- Pre-warm water bath to 42 °C
- Pre-warm LB medium
How it went:
Day 4
Materials required:
- Sub-culture DE3 in the morning.
- Need 1x5mL tubes per pair, plus two extra to be safe.
- Typical sub-culture: about 1.5 hours from 0.15 OD to early/mid-log.
- Last time starting batches at 12:10, 12:30 pm worked well (w/ lecture going till 1:45 pm), but test current crop of cells to double-check growth rate.
- Put calcium chloride (prep ~8 mL aliquots) on ice.
- LB+Amp/Cam plates
- LB broth, Amp, Cam
- Miniprep solution aliquots
- Sequencing primer thawed and diluted 1:100 TK CHECK/UPDATE
- Sterile DI water (200μL aliquots)
- Thaw NEB buffers and keep on ice, have enzymes at the ready.
- For digests: 12 μL parent IPC and 8 μL M124S miniprep for each group. TK UPDATE, FIGURE OUT AMOUNTS
- For sense of miniprep stock concentrations, see S10 Day 5 Talk W/F Yellow (E67K), S10 lane 3 in all gels, my notebook (T79P), Fahim's notebook (WT)
Day of Lab (T/W):
- Quiz (prepared by TA).
- Keep an eye on DE3 densities before and during lab.
- Prepare an aliquot each of M124S, T79P, and E67K to be sequenced at Genewiz, so we only need one set of alignment instructions for the students.
Days after Lab (W/R):
- Store plates in fridge, wrapped in parafilm.
- On T/W, pick two colonies per mutant to grow O/N in liquid culture (Amp+Cam).
- Also pick a positive control colony per student plate. (Or just three done by us, known to be correct.)
- Prepare DE3/WT-IPC in advance for all.
- Assuming will need to make 1:20 dilutions next time, need at least 2.4 mL of each
- Prep 3 (3 mL) O/N tubes to be on safe side
How it went:
- T/R
- W/F
Day 5
Materials required:
- Advance prep
- O/N cultures (see sub-culture notes below)
- Ice buckets out, per two teams to share.
- Part 1
- Put out LB for OD measurements, 10 mL per group plus a couple extra tubes.
- Sub-cultures
- Prepare each BL21 mutant candidate, 6 mL per tube.
- Prepare enough BL21-wild-type and BL21-reference mutants for each pair to have one tube as needed (plus make two extra of WT, and one of each reference mutant).
- In sum, there should be 4 tubes of BL21 per pair.
- In the past, ~1:20 dilution initiated between 10:00 and 10:30 am usually worked well. M124S grows somewhat more slowly.
- Thaw frozen IPTG or prepare fresh (0.1 M stock). IPTG MW = 238.3 g/mol = 0.095 g in 4 mL for 0.1 M. Put out 550 μL per two teams to share.
- Part 2
- Thaw digests
- One-half gel per group and ~ 500 mL TAE buffer per gel available.
- T/R: Three 1% gels and one 1.3% gel.
- W/F: Ideal is 2.5 1% gels and 1.5 1.3% gels.
- Loading dye out at teaching bench, about 4 aliquots (50 μL size) for those groups that did not yet add it on D4 and/or who forgot to prepare uncut samples.
- Check which groups didn't prepare uncut WT, have miniprep thawed and available in one or a few shared aliquots as needed.
- 1 Kbp and 100 bp ladders available. Two 100 μL aliquots of the 1 Kbp should suffice for both days.
- Put out sample sign. at gel bench as reminder.
- Part 4
- Colonies out at teaching bench
Day of Lab (R/F):
- Make sure students turn roller back on!
- Make sure students measure, then spin down and save at least their -IPTG samples.
- For recalcitrant +IPTG samples (no color change), continue induction at RT overnight.
Day after Lab (F/Sa):
- Measure the OD (1:10 dilution), then spin down and freeze any +IPTG samples cultured O/N.
- Post the OD values to the wiki.
How it went:
- S13
- T/R: Lots of samples growing quite slowly compared to previous years. Should start at 10 am instead of 10:30 am for W/F. Possible reasons: chloramphenicol made very fresh -- maybe lower slightly? Ditch antibiotics altogether?
- W/F:
S10
- Starting 1:20 sub-culture at 10:30 am (T/R) or even 11:15 am (W/F) gave high log ODs, 1.5 or 1, respectively.
- More than half the groups had green pellets after 2-2.5 hours of culture.
- M124S grows more slowly :( Most students had pale yellow pellet, some went with it, some grew O/N. After O/N growth, all pellets were quite green and large.
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
- BPER (3 mL aliquots, 30 μL 10% BSA and 30 μL protease inhibitors)
- 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:
- Pipetting reservoirs - 2 per group
- Calcium solutions - 0.5 mL/soln/group
Day of Lab:
- 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
- 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:
- 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.