Electrophoesis Gel Preparation
- 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.
First Stock Digest
Procedure
A) Get biobricks and enzymes from freezer.
SpeI, PstI, XbaI
The following list of biobricks are:
- KAH01 hPCD
- KAH03 flyPCD
- KAH04 fshPCD
- KAH204 mCh: SP1AB
- KAH205 mCh: SP1A
- KAH206 mCh: SP1B
- KAH225 mCh: p65
B)Follow digest procedure:
DNA 20.0 µL
Enzyme 1 1.0 µL
Enzyme 2 1.0 µL
10x buffer 3.0 µL
dH2O 5.0 uL
= 30.0 uL
 Prep for Assembly
Digest Order:
Biobrick Restriction Enzymes
- 1. KAH01 SpeI/PstI
- 2. KAH03 SpeI/PstI
- 3. KAH04 SpeI/PstI
- 4. KAH204 XbaI/PstI
- 5. KAH205 XbaI/PstI
- 6. KAH206 XbaI/PstI
- 7. KAH225 XbaI/PstI
C) Put reactions into heat block for 10 minutes at 37°C
Note: Dr. Haynes completed 1, 6 and 7.
Photos
 Digests 1-7
 Gel Cut Out
Next Steps
- DNA purification (QIAGEN) and then ligation.
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Engineering PC-TFs
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