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| '''DNase I Solution'''
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| 2.0 Units/ml DNase I
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| 50 mM Tris-HCl, pH 7.4
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| Prepare just before use
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| 10 mM MnCl2
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| '''Fragmentation'''
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| 1. Dissolve 2 to 5 μg of parent DNA in 10 μl of ddH2O (see Hint #1).
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| 2. Incubate the solution at 15 °C.
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| 3. Prepare 40 μl of DNase I solution and incubate at 15°C.
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| 4. Add 10 μl of the parent DNA solution to the 40 μl of DNase I solution at 15°C.
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| 5. Transfer 10 μl aliquots from the digestion reaction to individual tubes on ice containing 1 μl of 0.5 M EDTA after 1, 2, 3, 5, and 10 min of incubation. (see Hint #2).
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| 6. Electrophorese the samples on a 2% (w/v) Agarose gel (see Protocol ID #1265 and Hint #3) containing 0.5 μg/μl Ethidium Bromide (CAUTION! See Hint #9).
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| 7. Place the gel on a preparative UV illuminator (typically at a wavelength of 366 nm) to visualize the DNA (see Hint #4).
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| 8. Excise the region of the gel containing DNA of the desired molecular weight range (see Hint #5).
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| 9. Purify the DNA fragments from the Agarose gel with the QIAEX II gel extraction kit (Qiagen), or Wizard prep (Promega) according to the manufacturer's instructions.
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| 10. Elute the DNA fragments from the DNA binding resin with 10 μl of ddH2O.
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| '''Assembly'''
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| 1. Combine the following in a microcentrifuge tube:
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| 5 μl of 10X Taq Buffer
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| 5 μl of dNTP Mix
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| 10 μl of the purified fragments
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| 2.5 Units of Taq Polymerase
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| Add ddH2O to a total volume of 50 μl.
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| 2. Incubate the reaction for 3 min at 94°C.
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| 3. Amplify the DNA fragments in a thermocycler with the following program:
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| 30 sec at 94°C
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| 1 min at 55°C
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| 1 min at 72°C
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| Lengthen each extension step by an additional 5 sec per cycle.
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| Repeat for 40 cycles (see Hint #6).
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| 4. Visualize a small portion of the reaction by electrophoresis on a 2% (w/v) Agarose Gel (see Protocol ID#1265 and Hint #7).
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| '''Amplification'''
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| 1. Mix the following components in a microcentrifuge tube:
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| 10 μl of 10 X Taq Buffer
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| 10 μl of dNTP Mix
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| 0.5 μM each Primer (Primer #1 and Primer #2)
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| 1 μl of Assembly Reaction from Step #B3
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| 5 Units of Taq Polymerase
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| Add ddH2O to a total volume of 100 μl.
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| 2. Amplify the DNA fragments in a thermocycler with the following program:
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| 30 sec at 94°C
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| 30 sec at 55°C
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| 30 sec at 72°C
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| Repeat for 30 cycles (see Hint #6).
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| 3. Visualize a small portion of the reaction by electrophoresis on a 2% (w/v) Agarose Gel (see Protocol ID#1265 and Hint #8).
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| 4. Electrophorese the DNA on a 2% (w/v) preparative Agarose gel (see Protocol ID #1265 and Hint #3) containing 0.5 μg/μl Ethidium Bromide (CAUTION! See Hint #9) to purify the DNA sequences for cloning into an expression vector.
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| Adapted from http://www.bio.com/protocolstools/protocol.jhtml?id=p2189
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