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 Engineering PC-TFs
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3/18/22
Cutting inserts to be placed in mammalian vector
A) Get biobricks and enzymes from freezer.
SpeI, PstI, XbaI
The following list of parts are:
| Stock Digest
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
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Digest Order:
Biobrick Restriction Enzymes
- 1. BL01 XbaI/PstI
- 2. BL05 XbaI/PstI
- 3. BL09 XbaI/PstI
- 4 BL12 XbaI/PstI
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 45 min at 100 V. Check gel under UV light.
| Reagent
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Volume
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30 μL/lane, 1% agarose; Ladder
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| DNA (plasmid) |
15.0 μL
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| XbaI |
1.0 μL
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| PstI |
1.0 μL
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| Green 10x FastDigest buffer |
1.0 μL
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| dH2O |
10 μL
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30 μL --> 37°C/ ~10 min.
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| #
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Biobrick
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ng/µL
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| 1
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hPCD : mCh : SP1AB (BL01)
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18.834
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| 2
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fshPCD : mCh : SP1AB (BL05)
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14.471
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| 3
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flyPCD : mCh : SP1A (BL09)
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7.302
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| 4
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hPCD : BL01 (BL12)
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10.84
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Engineering PC-TFs
