Haynes Lab:Notebook/Engineering PC-TFs/2013/03/18

Current revision

Engineering PC-TFs

Cutting inserts to be placed in mammalian vector

A) Get biobricks and enzymes from freezer.

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

Digest Order:

Biobrick Restriction Enzymes

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.

@@ Line 27: / Line 27: @@
 *Enzyme 2     1.0  µL
 *10x buffer   3.0  µL
 *dH2O         5.0  uL
 *            = 30.0 uL </font>
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