20.109(S08): TA notes for module 1
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Revision as of 00:00, 28 December 2007 by AgiStachowiak (Talk)
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
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.
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.
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
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.