Contributor: |
The Laboratory of Andrew Murray at Harvard University
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This protocol can be used to sequence from a plasmid, use 50-fold excess of Primer 1 (2 μM) vs. Primer 2 (0.04 μM) to obtain a mainly single stranded product, then use the sequencing Primer and sequence directly without purifying the product. |
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1. Pick one yeast colony and resuspend it in 100 μl Reaction Mix in a 0.5 ml microcentrifuge tube.
2. Boil 5 min, centrifuge briefly and cool.
3. Add 0.5 μl Taq Polymerase, overlay each reaction tube with Mineral Oil
4. Amplify for 30 cycles:
94°C 1 min
42°C 2 min
65°C 4 min
Final extension at 65°C for 5 min
5. Run 10 to 15 μl on an Agarose gel (see protocol for Agarose Gel Electrophoresis of DNA).
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TE Buffer |
| 10 mM Tris pH 8.0 1 mM EDTA
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Chloroform/Isoamyl Alcohol |
| 25:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)
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Phenol/Chloroform |
| 1:1 Phenol:Chloroform (CAUTION! see Hint #1)
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Reaction Mix |
| 5 μl 2 μM 3' Primer 63.5 μl ddH2O 10 μl 10X PCR Buffer 16 μl dNTP's (1.25 mM dATP, dCTP, dGTP, dTTP) 5 μl 2 μM 5' Primer
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PCR Buffer (10X) |
| 15 mM MgCl2 500 mM KCl 100 mM Tris, pH 8.3
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Phenol Ammonium Acetate DNA Polymerase, Taq EDTA Ethanol Oligonucleotide Mineral Oil Tris Potassium Chloride dCTP dTTP dGTP dATP Magnesium Chloride Isoamyl Alcohol Chloroform
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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