October 11, 2013
Restriction Digest
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
|
Volume
|
 
30 μL/lane, 1% agarose; Ladder
|
| DNA (plasmid) |
20.0 μL
|
| XbaI |
1.0 μL
|
| PstI |
1.0 μL
|
| Green 10x FastDigest buffer |
3.0 μL
|
| dH2O |
5 μL
|
| |
30 μL --> 37°C/ ~10 min.
|
ZymocleanTM Gel DNA Recovery Kit
- Add 3 volumes of ADB Buffer to each volume of gel (600 μL).
- Incubate at 55°C for 10 minutes.
- Centrifuge at ≥ 10,000 x g for 30 seconds. Discard flow-through.
- Add 200 μL of DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat wash step.
- Place Zymoclean Column into a new 1.5 mL tube. Add 20μL of dH2O to elute DNA
| #
|
Part
|
ng/µL
|
| 1
|
CMV
|
0.851
|
| 2
|
CMV
|
15.535
|
|
