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| | | ||||1 rxn||x 4 | | | ||||1 rxn||x 4 |
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| - | | DNA plasmid||||3||-- | + | | DNA plasmid||3||-- |
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| - | | enzyme 1||||1||4 | + | | enzyme 1||1||4 |
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| - | | enzyme 2||||1||4 | + | | enzyme 2||1||4 |
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| - | | 10x buffer||||1.5||6 | + | | 10x buffer||1.5||6 |
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| - | | dH2O||||8.5||34 | + | | dH2O||8.5||34 |
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Revision as of 22:03, 2 March 2013
 Engineering PC-TFs
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3/2/2013
E/P Digest for FURI 2013-2014 application
- Retrieve BL01, BL05, BL09, BL011 assembly from freezer and thaw at room temperature.
- Also let restriction enzymes thaw.
- Do restriction digest (EcoRI/PstI)Diagnostic digest
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| 15 µl Total
| Master Mix
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| | | 1 rxn | x 4
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| DNA plasmid | 3 | --
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| enzyme 1 | 1 | 4
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| enzyme 2 | 1 | 4
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| 10x buffer | 1.5 | 6
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| dH2O | 8.5 | 34
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- Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
- Place each tube in heat block 37°C.
Follow steps for gel electrophoresis.
- Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
- Fill gel flask with up to 60 ml of TA buffer.
- Create 1% gel by putting .6 grams of agarose into flask.
- Microwave agarose solution for 40 seconds
- Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
- When flask is taken out of microwave, make sure that the agarose is completed dissolved.
- Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
- Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
- Pour gel into tray.
- Wash the agarose gel flask.
- Run gel for 30 min at 100 V. Check gel under UV light.
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Engineering PC-TFs
