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:

1. Put out LB for OD measurements, few mL per group.
2. Sub-culture each DE3/mutant, 6 mL per tube.
   • Last year, ~1:20 dilution initiated between 10:00 and 10:30 am worked well.
   • This means 4 tubes of mutants per pair.
3. Also sub-culture enough DE3/wild-type and DE3/M124S/E67K/T79P for each pair to have one tube as needed (plus make two extra).
4. 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.
5. One-half gel per group, TAE buffer, 1 Kbp and 100 bp ladders available. Put out sample sign.

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:

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:

1. Cell lysis
   • BPER (4 mL aliquots, +40 μL 10% BSA and inhibitors)
   • Lysis enzyme available on ice up front
   • Water (150 μL aliquots)
2. 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
3. SDS-PAGE, ready in hood
   • 2X sample buffer (add 5% β-Me at last minute)
   • Water baths with boiling chips, turn early on in lab
   • Lid locks
   • Waste bottle for stain
4. Protein purification (see googledoc)
   • Note: prepare solutions ~15% in excess of needed volume
   • Water, Charge Buffer (actually, will probably will buy pre-charged resin this year)
   • Binding Buffer, Wash Buffer, and Elution Buffer w/protease inhibitors
   • Small BSA aliquots ready.
5. Protein concentration
   • Have 5X Coomassie stain from Bio-Rad, water, tubes ready

Day of Lab:

• No quiz - a very busy day!
• 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:

• 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.