Difference between revisions of "20.109(S08): TA notes for module 2"
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*Pick one colony per mutant to grow O/N in liquid culture (Amp+Cam). | *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. | **Use either the 1X or 10X dilution of cells, depending which has independently accessible colonies. | ||
+ | |||
+ | ===[[20.109(S08):Induce protein expression (Day4) | Day 4]]=== | ||
+ | |||
+ | '''Materials required:''' | ||
+ | |||
+ | *Sub-culture each DE3/mutant, 5 mL per 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. |
Revision as of 20:21, 3 January 2008
General notes
Key preparation:
- Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.
Daily Notes
Day 1
Materials required:
- None: all work today is computer work.
Day of Lab:
- No quiz.
- Primers for mutagenesis must be ordered right away!
Day 2
Materials required:
- Quick-Change SDM kit. ?1/2? 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.
- sterile DI water
- LB+Amp plates (at least 15 per day)
- autoclaved glass tubes (at least 25 per day)
- LB broth, ampicillin
- 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=...) O/N cultures of DE3 cells, to be sub-cultured tomorrow.
Day 3
Materials required:
- 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.
- Put calcium chloride (prep 0.5 mL aliquots) on ice.
- LB+Amp/Cam plates (~70 per day)
- Calcium titration solutions
- Sequencing primers thawed
- 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.
Day 4
Materials required:
- Sub-culture each DE3/mutant, 5 mL per 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.