20.109(S13): TA notes for module 2

Long overdue for a revision

General notes

Key preparation:

• Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
• Mutant IPC plasmid (M124S, E67K, and T79P) should also be prepared in advance.
• 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 two protein mutants, and test two candidate colonies per mutant. Specifically, students will choose only one mutation of their own, and run a 'positive control' mutation in parallel.

Daily Notes

Day 1

Materials required:

1. None: all work today is computer work.

Day of Lab (T/W):

• No quiz.
• Primers for mutagenesis must be ordered right away, rush delivery!
• Enzymes for diagnostics may need to be ordered as well, check designs.

How it went:

• Two odd issues cropped up:
  • In fact, compared to most CaM sequences, two base pairs are missing in the IPC CaM. The Met is easily understandable, the other throws students off more.
  • Watcut link should be strictly checked and instructions should be included for each pair to name their primer something unique. Sometimes old primers or incomplete enzyme lists were automatically used.
• Also, instructions to re-check primer Tm should probably be in bold or even emphasized during pre-lab. I think many folks skipped that step.

Day 2

Materials required:

1. RT water for primer resuspension
2. Quick-Change SDM kit. 1 reaction per student, plus 1 control reaction, plus spare reagents (ideally).
   • cat # 200519

Day of Lab (T/W):

• Quiz (prepared by TA)
• Prepare PCR tubes on racks obtained from the freezer
• Get primers (forward and reverse) ready just before lab lecture ends
• Prepare a few aliquots of Master Mix for students, plus a control reaction:
  • Each mutagenesis reaction should have 5 μL of buffer, 1 μL of dNTPs, and 37 μL of water, for a total of 43 μL.
  • See googledoc for total calculations
  • Control rxn. is: 43 μL Master Mix, 2 μL DNA, 1.25 μL each primer, and thus 2.5 μL extra water.
• Guide journal article discussion (assign figures at beginning of class).
• At end of day: freeze SDM DNA

Day 3

Materials required:

1. Agarose gel electrophoresis
   • 1% agarose gels, all groups can fit on one gel
   • TAE buffer
   • 1 Kb ladder
   • Post sample table at gel bench, put out nitrile gloves
2. Bacterial transformation
   • LB+Amp plates - > 1.5 l of LB requires 45 min of autoclave and is hard to pour, should separate into 2 flasks for 30 min of autoclave
   • autoclaved glass tubes
   • super-competent XL1-Blue cells (come with SDM kit, plus buy extra for negative controls)

Day of Lab (R/F)

• Pre-warm water bath to 42 C, tube racks, etc.
• Teaching faculty should prepare one positive control plate (in addition to the ones made by the students).

Days after Lab:

• Check student plates for colonies next day.
• During spring break, check a few colonies. Then, pluck two colonies per mutant plate day before next lab, and grow liquid O/N cultures. (Amp only, no Cam yet!)
• Students with no colonies will be given their choice of any student candidate. (Some check on repeatability this way.)

How it went:

• About half the groups got lots of colonies, three more groups got just a few colonies, and several groups got no colonies.

Day 4

Materials required:

1. 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.
2. Put calcium chloride (prep ~8 mL aliquots) on ice.
3. LB+Amp/Cam plates
4. LB broth, Amp, Cam
5. Miniprep solution aliquots
6. Sequencing primer thawed and diluted 1:100 TK CHECK/UPDATE
7. Sterile DI water (200μL aliquots)
8. Thaw NEB buffers and keep on ice, have enzymes at the ready.
9. 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
• 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.