20.109(S08): TA notes for module 2

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.

Scheme: each pair of students will make two protein mutants, and test two candidates per mutant.

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!

Day 2

Materials required:

1. Quick-Change SDM kit. 2 reactions per student, plus 1 control reaction, plus spare reagents (ideally).
   • cat # 200519
   • e.g., for 12 pairs and 1 mutant per pair, two 10 rxn kits would suffice; for 12 pairs and 2 mutants per pair, 1 30 rxn. kit should suffice.
2. sterile DI water (>1mL cold, 1.5 mL RT/group)
3. LB+Amp plates (at least 15 per day)
4. autoclaved glass tubes (at least 25 per day)
5. LB broth (>72mL/day)
6. 1000X ampicillin - central stock per day (>72uL/day)
7. competent XL1-Blue cells (come with SDM kit) -- for us to do transformation after class

Day of Lab (R/F):

• Aliquot SDM reagents and prepare Master Mix for students:
  • Each mutagenesis reaction should have 5 μL of buffer, 1 μL of dNTPs, and 37 μL of water, for a total of 43 μL.
  • Students will receive 43*2 + 10% excess = 95 μL of Master Mix each. Thus, per day we need ~ 95*6*15%excess = 650 μL
  • Mix together: 76 μL rxn. buffer, 15 μL dNTPs, and 564 μL water on each day.
    • This is slightly more than a 30 rxn. kit, and thus the old 10 rxn. kit should be dipped into for the excess.
  • Control rxn. is: 43 μL Master Mix, 2 μL DNA, 1.25 μL each primer, and thus 2.5 μL extra water.
• Set up Part 3 (liquid culture prep) as a station on the TA bench.
• Quiz (prepared by TA).
• Guide journal article discussion (assign figures at beginning of class).
• At end of day: transform mutant DNA, after digesting parental plasmid, into competent XL1-Blue cells.

Pre-warm water bath to 42 C, tube racks, etc.

Day after Lab (F/Sa):

• Ensure that positive control (pWhitescript) produced colonies.
• Pluck two colonies per mutant plate, and grow liquid O/N cultures. (Amp only, no Cam yet!)
  • Students with no colonies will be given my pilot minipreps (Y64D, etc.) next time.
• On Sa/Su, prepare minipreps from each candidate (4x6 = 24 per day).
• On M/T, grow several (N=14x3mL -- or 7x6mL and split?) O/N cultures of DE3 cells, to be sub-cultured on Day 3 am.
• On M (at latest), streak out DE3 carrying wild-type IPC on a fresh Amp/Cam plate. (likely do this earlier and use to prep the wild-type IPC needed for Day 3, a way to test that the cells are still carrying the right plasmid in any case)
  • Done earlier, and did not get fluorescent cell pellet. Prepared fresh transformants, and got the right protein this time. (May have also been due to being at slightly higher OD, closer to 0.5 than 0.4, but hard to say for sure which mattered more).

How it went:

• Section 1, post-class prep from 5:00-7:30
• Section 2, post-class prep from 4:45-7:00

Day 3

Materials required:

1. Sub-culture DE3 in the morning.
   • Need 2x3mL tubes per pair, or ~ 14 tubes total to be safe.
   • Typical sub-culture: from OD > 2 to OD = 0.1 may take ~3-5 hours to reach OD = 0.6. Sub-culture to 0.15 or even 0.2 makes for shorter and more predictable lag phase - go with that! Stagger tubes a bit and test after 2-3 h to be safe.
     • So start some at 8:30, 9, 9:30 perhaps?
2. Put calcium chloride (prep ~8 mL aliquots) on ice.
3. LB+Amp/Cam plates (~70 per day)
4. LB broth, Amp, Cam
5. Calcium titration solutions
   • per pair, ~ 50μL of EB blanking solution
   • per pair, ~ 20μL of each calcium solution (probably should be shifted slightly from previous values, not catching enough data near the inflection point)
   • per pair, ~150 μL of wild-type protein solution (advance prep!)
6. Sequencing primers thawed and diliuted 1:100
7. Sterile DI water (200μL aliquots)

Day of Lab (T/W):

• Quiz (prepared by TA).
• Keep an eye on DE3 densities before and during lab.

Day after Lab (W/R):

• Pick two colonies per mutant to grow O/N in liquid culture (Amp+Cam).
  • Use either the 1X or 10X dilution of cells, depending which has independently accessible colonies.
• Also pluck DE3/WT-IPC (8x6mL)

How it went:

• Both days, DE3 grew quite fast. Starting one batch at 12:10 pm and another at 12:30 pm worked out well.
• Lecture lasted till 1:40/45ish, so cells grew ~ 1.5 hrs.

Day 4

Materials required:

1. Sub-culture each DE3/mutant, 6 mL per tube.
   • As before, ~1:20 dilution initiated between 8:30 and 9:30 am should work well.
   • This means 4 tubes per pair, = 24 per day, and the 6 (+ couple extra) below.
2. Also sub-culture enough DE3/wild-type for each pair to have one tube.
3. Thaw frozen IPTG or prepare fresh (0.1 M stock).

Day of Lab (R/F):

• Likely no quiz (especially if we can get sequencing results in time), or a very easy one.
• Make sure students measure, then spin down and save at least their -IPTG samples.
• For recalcitrant +IPTG samples (no colour 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.
  • Email the OD values to appropriate students and/or post to wiki.

How it went:

• WT and most mutants grew well if started b/w 10 and 10:30 am.
• Lecture lasted till 1:30/35, short quiz was used to move things along.

Day 5

Materials required:

1. Cell lysis
   • BPER (4 mL aliquots, +40 μL 10% BSA and inhibitors)
2. SDS-PAGE
   • Polyacrylamide gels (1 per pair).
   • TGS buffer
   • 2X sample buffer (142.5 μL aliquots, add 7.5 μL β-Me last-minute)
   • Water (150 μL aliquots)
3. Protein purification (multiply recipes by 6 pairs per day)
   • Note: each solution contains ~20% in excess of needed volume
   • Water: ~ 3 mL per pair
   • Charge Buffer: 4.4 mL aliquot per pair (550 μL 8X stock, ~3.85 mL water) = 3.3/23.1 mL
   • Binding Buffer: 9.4 mL aliquot per pair (1.175 μL 8X stock, 94 μL inhibitor, ~8.15 mL water) = 48.9/7.05/0.564 mL
   • Wash Buffer: 4.4 mL aliquot per pair (550 μL 8X stock, 44 μL inhibitor, ~3.8 mL water) = 3.3/22.8/0.264 mL
   • Elute Buffer: 3.6 mL aliquot per pair (900 μL 4X stock, 36 μL inhibitor, ~2.65 mL water) = 5.4/15.9/0.216 mL
4. 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 6 platereader use.

Day 6

Materials required:

1. Pipetting reservoirs - 12
2. Calcium solutions - 5 mL per solution

Day of Lab:

• Quiz (prepared by TA).
• Post data to wiki.

Day 7

Materials required:

1. None: all computer work today.

Day of Lab:

• Quiz (prepared by TA).