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.

Note: amounts still need to be worked out a bit. Whether we do 1 or 2 mutants per pair of students depends on 1) whether we can get sequencing data guaranteed in 44h, 2) whether purifying 1 mL of protein per pellet (instead of 0.5 mL) will give bright enough fluorescence, or if we need to have the students purify two pellet per sample in order to get replicate data

Daily Notes

Day 1

Materials required:

1. None: all work today is computer work.

Day of Lab:

• No quiz.
• Primers for mutagenesis must be ordered right away!

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

Day of Lab:

• 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:

• Ensure that positive control (pWhitescript) produced colonies.
• Pluck two colonies per mutant plate, and grow liquid O/N cultures. (Amp only, no Cam yet!)
• Grow several (N=36x3mL -- or 18x6mL and split?) O/N cultures of DE3 cells, to be sub-cultured tomorrow.

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.
2. Put calcium chloride (prep 0.5 mL aliquots) on ice.
3. LB+Amp/Cam plates (~70 per day)
4. Calcium titration solutions
5. Sequencing primers thawed
6. Sterile DI water

Day of Lab:

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

Day after Lab:

• Pick one colony 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 grow DE3/wild-type (AMOUNT)

Day 4

Materials required:

• Sub-culture each DE3/mutant, 5 mL per tube.
• Also sub-culture enough DE3/wild-type for each pair to have one tube.
• Thaw frozen IPTG or prepare fresh (0.1 M stock).

Day of Lab:

• Likely no quiz (especially if we can get sequencing results in time).
• Make sure students measure, then spin down and save at least their -IPTG samples.
• For recalcitrant +IPTG samples, continue induction at RT overnight.

Day after Lab:

• Measure the OD (1:10 dilution), then spin down and freeze any +IPTG samples cultured O/N.
  • Email the OD values to appropriate students.

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 - N
2. Calcium solutions - N 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).