Haynes Lab:Notebook/Engineering PC-TFs/2013/03/03: Difference between revisions
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==Summary== | ==Summary== | ||
<font size=3>''E/P Digest for FURI 2013-2014 application''</font> | |||
* 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'' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''15 µl Total''' | |||
| align="center" style="background:#f0f0f0;"|'''Master Mix''' | |||
|- | |||
| ||1 rxn||x 4 | |||
|- | |||
| DNA plasmid||3||-- | |||
|- | |||
| enzyme 1||1||4 | |||
|- | |||
| enzyme 2||1||4 | |||
|- | |||
| 10x buffer||1.5||6 | |||
|- | |||
| dH2O||8.5||34 | |||
|- | |||
| | |||
|} | |||
*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. | |||
* Label Tubes | * Label Tubes |
Revision as of 22:43, 3 March 2013
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SummaryE/P Digest for FURI 2013-2014 application
Follow steps for gel electrophoresis.
1. BL01 2. BL05 3. BL09
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