Difference between revisions of "20.109(S08): TA notes for module 2"
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'''Materials required:''' | '''Materials required:''' | ||
− | #Sub-culture each DE3/mutant, | + | #Sub-culture each DE3/mutant, 6 mL per tube. |
#Also sub-culture enough DE3/wild-type for each pair to have one 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). | #Thaw frozen IPTG or prepare fresh (0.1 M stock). |
Revision as of 15:25, 7 March 2008
Contents
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:
- 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:
- 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.
- 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 (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:
- 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?
- Put calcium chloride (prep 0.5 mL aliquots) on ice.
- LB+Amp/Cam plates (~70 per day)
- LB broth, Amp, Cam
- Calcium titration solutions
- per pair, ~ 50μL of EB blanking solution
- per pair, ~ 20μL of each calcium solution
- per pair, ~150 μL of wild-type protein solution (advance prep!)
- Sequencing primers thawed and diliuted 1:100
- 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:
- Sub-culture each DE3/mutant, 6 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), or a very easy one.
- 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 and/or post to wiki.
Day 5
Materials required:
- Cell lysis
- SDS-PAGE
- Polyacrylamide gels (1 per pair).
- TGS buffer
- Sample buffer
- 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)
- 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:
- Pipetting reservoirs - 12
- Calcium solutions - 5 mL per solution
Day of Lab:
- Quiz (prepared by TA).
- Post data to wiki.
Day 7
Materials required:
- None: all computer work today.
Day of Lab:
- Quiz (prepared by TA).