Team:WashU/Protocols/LargeDoubleDigestion

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==Large Scale Double Digest==
==Large Scale Double Digest==
'''Supplies'''<br>
'''Supplies'''<br>
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1. 2.5ng DNA of part to be digested<br>
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1. 2.5micrograms DNA of part to be digested<br>
2. 3ul 1st Enzyme<br>
2. 3ul 1st Enzyme<br>
3. 3ul 2nd Enzyme<br>
3. 3ul 2nd Enzyme<br>
4. 5ul 10x Buffer for enzymes<br>
4. 5ul 10x Buffer for enzymes<br>
5. 0.5ul BSA if required for enzymes<br>
5. 0.5ul BSA if required for enzymes<br>
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5. ddH20 to bring total volume to 50ul<br>
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6. ddH20 to bring total volume to 50ul<br>
'''Procedure'''<br>
'''Procedure'''<br>
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3. Using a water bath or thermo cycler, incubate mixture at 37C for 2hrs. <br>
3. Using a water bath or thermo cycler, incubate mixture at 37C for 2hrs. <br>
4. Heat inactivate restriction enzymes by heating mixture at 80C for 20min (or 65C depending upon enzymes used)<br>
4. Heat inactivate restriction enzymes by heating mixture at 80C for 20min (or 65C depending upon enzymes used)<br>
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[https://2012.igem.org/Team:WashU/Protocols Back to Protocols]

Latest revision as of 21:31, 9 August 2012


Large Scale Double Digest

Supplies
1. 2.5micrograms DNA of part to be digested
2. 3ul 1st Enzyme
3. 3ul 2nd Enzyme
4. 5ul 10x Buffer for enzymes
5. 0.5ul BSA if required for enzymes
6. ddH20 to bring total volume to 50ul

Procedure
1. Mix water, DNA, buffer and BSA (if required) in a 200ul PCR tube, vortex to mix.
2. Add both enzymes and vortex again to mix.
3. Using a water bath or thermo cycler, incubate mixture at 37C for 2hrs.
4. Heat inactivate restriction enzymes by heating mixture at 80C for 20min (or 65C depending upon enzymes used)


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