Haynes Lab:Notebook/Engineering PC-TFs/2013/03/02: Difference between revisions
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==Summary== | ==Summary== | ||
* | * Retrieve mini preps for flyPCD assembly from freezer and thaw at room temperature. | ||
* Also let restriction enzymes thaw. | |||
*Do restriction digest (EcoRI/PstI)''Diagnostic digest'' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''15 µl Total''' | |||
| align="center" style="background:#f0f0f0;"|'''Master Mix''' | |||
|- | |||
| ||||1 rxn||x 9 | |||
|- | |||
| DNA plasmid||||3||-- | |||
|- | |||
| enzyme 1||||1||9 | |||
|- | |||
| enzyme 2||||1||9 | |||
|- | |||
| 10x buffer||||1.5||13.5 | |||
|- | |||
| dH2O||||8.5||76.5 | |||
|- | |||
| | |||
|} | |||
*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. | |||
Revision as of 18:59, 2 March 2013
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Summary
Follow steps for gel electrophoresis.
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