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
3. LB+Amp plates (at least 15 per day)
4. autoclaved glass tubes (at least 25 per day)
5. LB broth, ampicillin
6. competent XL1-Blue cells (come with SDM kit)

Day of Lab (R/F):

• Aliquot SDM reagents and prepare Master Mix for students:
• 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.

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!)
• 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)

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. 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 0.5 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)

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.

Day 5

Materials required:

1. Cell lysis
2. SDS-PAGE
   • Polyacrylamide gels (1 per pair).
   • TGS buffer
   • Sample buffer
3. Protein purification (multiply recipes by 6 pairs per day)
   • Note: each solution contains ~20% in excess of needed volume
   • Charge Buffer: 4.4 mL aliquot per pair (550 μL 8X stock, ~3.85 mL water)
   • Binding Buffer: 9.4 mL aliquot per pair (1.175 μL 8X stock, 94 μL inhibitor, ~8.15 mL water)
   • Wash Buffer: 4.4 mL aliquot per pair (550 μL 8X stock, 44 μL inhibitor, ~3.8 mL water)
   • Elute Buffer: 3.6 mL aliquot per pair (900 μL 4X stock, 36 μL inhibitor, ~2.65 mL water)
4. Protein concentration

Day of Lab:

• No quiz - a very busy day!
• Transfer gels to fresh destain buffer.
• Collect all purified protein samples from students and store at 4 °C.

Day after Lab:

• Transfer gels to water and take pictures once they swell up a bit.
• 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).