Difference between revisions of "20.109(S08): TA notes for module 1"
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− | 94C 1min | + | 94C 1min; 55C 1min; 72C 1min; (Repeat 35 times) |
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− | 72C 1min | + | |
− | (Repeat 35 times) | + | |
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− | 72C 10min | + | 72C 10min; |
4C hold forever | 4C hold forever | ||
Revision as of 19:24, 8 January 2008
Contents
General notes
Key preparation:
- In case something goes wrong, spare PCR product should be prepared by TA prior to term.
- A plan for preparing the high volume of cell culture must be determined well in advance.
Plasmids used:
- pCX-EGFP:
- contains full-length EGFP gene
- Amp resistant
- f1 ori
- SV40 ori
- pCX-NNX:
- a mock version of pCX-EGFP, contains no EGFP gene
- Amp resistant
- f1 ori
- some altered restriction sites compared to pCX-EGFP, and SV40 ori deleted
- pCX-EGFP with 3' deletion - still have this in lab, how labeled?
Daily Notes
Day 1
Materials required:
- PCR Master Mix (2.5X), ~ 50 μL per group
- DI water, prep 100 μL aliquot per group
- 5 μL aliquots each of pCX-EGFP, D32N-fwd, D32N-rev
- 2 PCR tubes per group
Keep everything cold!
Day of Lab:
- Lab orientation quiz (not a TA quiz) will be taken today.
- Remember to freeze PCR products when they are ready.
Instructor's Bench
- Ice bucket
- PCR Master Mix (taken out of -20C midway through labtime)
- Sterile H20 in 15mL Falcon
- Primters for PCR (D32N-fwd: NO47, D32N-rev: NO48, 100 pmol/ul)
- Template for PCR (pCX-EGFP 100 ng/ul)
- Beaker with PCR tubes
Prep Work
pCX-EGFP has to be prepared from the glycerol stock. The bacterial stock is NBXXX and a few Qiagen minipreps should be enough for the module (some will also be needed later as a transfection control). You should determine the concentration of the stock. For PCR, dilute stock to about 100 ng/ul.
Other DNA samples that will be needed later in the module come from:
- NBXXXX pCX-NNX
- NB153 pCX-D32N (Xba/RI)
- NB168 pCX-D3G+Linker -- This plasmid needs to be cut in three ways and each bkb purified!
Check the PCR machine has proper protocol under NK1: 94C 4min
94C 1min; 55C 1min; 72C 1min; (Repeat 35 times)
72C 10min; 4C hold forever
Day 2
Materials required:
- Qiagen QIAquick PCR purification kit
- order # 28104, 50 rxns
- 1 rxn per group
- pCX-NNX, 10 μL per group
- NEB2 Buffer (~5 μL used per group)
- EcoRI and XbaI enzymes (~2 μL used per group)
Day of Lab:
- Quiz (prepared by TA).
- Thaw PCR products and template on ice.
Day 3
Materials required:
- Qiagen QIAquick gel extraction kit
- 2 extractions per group
- order # 28704, 50 rxns
- isopropanol (prep 500 μL aliquots)
- sterile DI water (prep 500 μL aliquots)
- loading dye for agarose gel electrophoresis (prep 35 μL aliquots)
- 1% agarose gels prepared in 0.5X TBE buffer
- each group has 10 samples, so requires 10 wells
- prepare 1 gel per two groups, but with 2 combs
- also prepare 1 more gel (2 combs!), for running purified products
- Single-enzyme digests - may be prepared on Day 2.
- pCX-NNX, XbaI cut only
- pCX-NNX, EcoRI cut only
- large (400 μL) and small (25 μL) volume reactions should be done for each of the above
Day of Lab:
- Quiz (prepared by TA).
- Run purified products (2 per group) on an agarose gel at the end of the day - post photograph.
- Freeze DNA at end of day.
Day 4
Materials required:
- Retrieve at last minute and keep on cold rack: ligation buffer, T4 ligase
- 3 M sodium acetate (prep 100 μL aliquots)
- Yeast tRNA (prep 25 μL aliquots)
- Cold 100% ethanol (prep 1 mL aliquots)
- Cold 70% ethanol (prep 3 mL aliquots)
- Sterile DI water (prep 100 μL aliquots)
- pCX-EGFP (1 μL/50 ng per group)
- competent cells
- XL1-Blue from Stratagene, cat # ?200249?
- 1 tube per group
- LB+Amp plates, 5 per group + spares
- VWR cat. #
- may get ~ 40 plates/Liter LB
- LB liquid medium
Day of Lab:
- Quiz (prepared by TA).
- Keep reagents for ligation reactions cold, make available to students as needed.
- Demonstrate and supervise bacterial transformation protocol.
- Transform cells with pCX-NNX to get fresh colonies.
- May be done a week or so in advance, if desired.
- Can also just try streaking frozen stock, if available.
- Tomorrow pick colonies for inoculations - 3 candidates per group, plus 1 pCX-NNX.
Day 5
Materials required:
- Liquid cultures (3 candidates, 1 pCX-NNX control per group).
- Miniprep solutions
- Soln I (prep 400 μL aliquots)
- Soln II components (prep 600 μL aliquots, each)
- Soln III (prep 800 μL aliquots)
- 100 % ethanol (prep 5 mL aliquots)
- 70 % ethanol (prep 3 mL aliquots)
- Sterile DI water (prep 250 μL aliquots)
- Digests: have all 4 NEB buffers and many restriction enzymes available
Day of Lab:
- Quiz (prepared by TA).
- Ensure DNA is frozen at end of day.
Day 6
Long-term preparation:
- Per student, one 60 mm dish of MES cells near confluence
Materials required:
- Agarose gels
- Half of class at a time will be in TC, so can't prepare just 1 gel per two groups
- Probably want to prepare 4 gels per 6 groups, but can prepare 1 per group for simplicity
- TC reagents
- Aliquot each at 10-20% excess
- PBS (12 mL per group)
- autoclaved gelatin (6 mL per group)
- Trypsin (1 mL per group)
- J1 medium(three tubes per group with precisely 10 mL each)
Day of Lab:
- Quiz (prepared by TA).
Day 7
Long-term preparation:
- Per group, one 24-well plate with 16 seeded wells.
- Seed with 105 cells 24 hrs prior to use.
- Need plasmid with EGFP truncated at 3' end.
Materials required:
- Lipofectamine (prep 50 μL aliquots)
- OptiMEM (prep 2 mL aliquots)
- TC reagents
- PBS (10 mL per group)
- T'xn Medium (10 mL per group)
Day of Lab:
- Quiz (prepared by TA).
Day 8
Materials required:
- TC reagents
- PBS (20 mL per group)
- Trypsin (4 mL per group)
- OptiMEM (2 mL per group) - 100 μL seems low for FACS
- FACS tubes
- 16 per group, plus extras
- BD Falcon tubes only, VWR cat # 60819-310
Day of Lab:
- No quiz.
- TA will run flow cytometer, and faculty will guide student preparations in the lab, or vice-versa.
Recipes/Reagents
Agarose Gel
- DNA gel: 1% agarose gel in 1X TAE, 1 g agarose, 100mL 1X TAE, 2 μL EtBr (wear nitrile gloves when handling EtBr!)
- 0.5X TBE insteadof 1X TAE for this lab... ?
- Loading dye for agarose gel: 250 μL 1% XC (xylene cyanol), 750 μL 40% glycerol, 10 μL RNase. Store at RT.
- 1kb marker: 10 μL 1kb marker stock (in -20 °C freezer), 10 μL loading dye, 90 μL H20
Bacterial growth media
- LB: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl per liter. 20g of Agar for plates. Autoclave 30 minutes with stirbar. Pour when ~55°. Let plates dry ON on bench and store in sleeves in 4°. For LB-Amp plates, add the Amp after autoclaving, once the mixture has cooled down.
- Amp: 100 mg/ml in H20. Filter and store at 4°. Use at 1:1000 in liq media. 2ml/L in plates
- Top Agar: 10 g Tryptone, 5 g Yeast Extract, 10 g NaCl, 1 g MgCl2*6H20 7 g Agar per liter. Autoclave then aliquot to 50 ml conical tubes or bottles. Store at RT. Melt in microwave in beaker of water, 2’ then keep molten in 55° water bath.
DNA Miniprep
- Soln I for miniprep: 2.3 ml 40% glucose, 2.5 ml 1M Tris 8, 2 ml 0.5M EDTA. To 100 ml with good H20. Store at RT
- Soln II for miniprep: equal parts 2% SDS (2g/100 ml H20): 0.4M NaOH (1.6g/100 ml H20). Store components at RT. Mix just enough just before using.
- Soln III for miniprep: 29.4 g KAc dissolved in 60 ml H20. Add 11.5 ml glacial acetic acid. Bring to 100 ml final volume. Store at RT.
Mammalian cell culture
- JI Growth Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml P/S/G, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF. Store 4°C
- Pre-Transformation Media: 500 ml DMEM (high glucose), 50 ml FBS (Atlanta Biologic, Inc.), 5 ml 100XG, 1 ml BME, 5 ml NEAA. Filter then add 50 ul LIF.
- J1 culture medium
- 100 U/ml Pen/Strep
- 0.3 mg/ml glutamine
- 0.1 mM BME,
- 1 mM Nonessential amino acids
- 10% serum
- LIF
- Sterile filter final mixture (0.2 mu;m filter)
- Gelatin
- 0.1% TC-grade gelatin prepared in H2O
- Autoclave mixture and allow to cool before use