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
| '
| '
| 15 µl Total
| Master Mix
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| | | 1 rxn | x 9
|
| DNA plasmid | | 3 | --
|
| enzyme 1 | | 1 | 9
|
| enzyme 2 | | 1 | 9
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| 10x buffer | | 1.5 | 13.5
|
| dH2O | | 8.5 | 76.5
|
|
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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.
Attempted to prepare gel in tray. Unforunately, this ended up being a struggle. I messed up setting up the wells twice and this caused me to not complete my diagnostic digest.
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Engineering PC-TFs
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