September 26, 2013
Restriction Digest
- Perform restriction digest for CMV promoter
Reagent
|
Volume
|
DNA (plasmid) |
10 μL
|
XbaI |
1.0 μL
|
PstI |
1.0 μL
|
Green 10x FastDigest buffer |
3.0 μL
|
dH2O |
15 μL
|
|
30 μL --> 37°C/ ~10 min.
|
- Measure the following into 1.5 mL tubes.
Reagent
|
15%
|
10%
|
7%
|
5%
|
2%
|
30% PEG |
50 μL |
33 μL |
23 μL |
17 μL |
7 μL
|
DNA |
15μL |
15 μL |
15 μL |
15 μL |
15 μL
|
dH2O |
35 μL |
52 μL |
62 μL |
68 μL |
78 μL
|
Total |
100 μL |
100 μL |
100 μL |
100 μL |
100 μL
|
- Centrifuge for 15 minutes at 10,000g.
DNA Clean & Concentrator
- Although there was no pellet, ran a DNA Clean & Concentrator on samples.
- Add 2 volumes of DNA Binder Buffer to each DNA sample (200 μL).
- Place mixture in spin column into 2 ml collection tube.
- Centrifuge at full speed for 30 seconds.
- Add 20 μL of DNA Wash Buffer and centrifuge for 30 seconds. Repeat this step.
- Place Zymo-Spin Column into a new 1.5 ml tube. Add 20 μL of water directly to column matrix and spin to elute DNA.
Gel Electrophoresis
- 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.
|