Lecture Video
Materials
- Agarose
- Ethidium bromide staining solution (0.5 μg/mL ethidium bromide in TBE buffer)
- DNA loading dye (Sigma, G2526)
- 0.5X TBE reservoir buffer (0.4 M TRIS-borate, 0.01 M EDTA, pH 8.0)
- Eco RI (100 units/μL) (New England Biolabs)
- Ava I (10 units/μL) (New England Biolabs)
- Hinc II (10 units/μl) (New England Biolabs)
- Rsa I (10 units/μL) (New England Biolabs)
- 10X NEB CutSmart Buffer (1X is 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM Magnesium Acetate, 1 mM Dithiothreitol, pH = 7.9 @ 25°C) (New England Biolabs)
- BSA (10 mg/ml) (New England Biolabs)
- DNase-free water
- Plasmid DNA isolated from E.coli (X ng/μL, concentration determined last week)
- Reference DNA: 1 kb Plus DNA reference ladder solution (New England Biolabs, N0550S) (1 μg for 5 μL load)
- Vortexer
- Microcentrifuge tube spinner
- Horizontal gel electrophoresis system with power supply
- UVP BioSpectrum 500 Imaging System (configured for 302 nm transillumination)
Restriction Digests
1. Ideally, you will want to load around 200 ng of digested DNA to get good bands on a gel. Using your your measured DNA concentation, calculate the volume of DNA that will give 200 ng.
2. You will prepare one restriction digest reaction that will contribute to the class data. We will decide who will carry-out which reaction in class. However, you should be prepared to make either a single or double digest of your DNA sample. Plan one of each reaction as shown in the table below. Use X (the DNA volume you calculated in step 1 to determine the amount of DNA and water to add. If your calculated volume exceeds the maximum volume, then simply use the maximum volume and do not add water.
| Component |
Single digests (μL) |
Double digests (μL) |
|
10X CutSmart buffer |
1 |
1 |
|
DNase-free DI H2O |
8 - X |
7 - X |
|
Plasmid DNA |
X (up to 8 μL) |
X (up to 7 μL) |
|
Enzyme 1 |
1 |
1 |
|
Enzyme 2 |
-- |
1 |
|
Total |
10 |
10 |
3. Add the components in the order shown in the above chart. All enzyme solutions will be on ice in a desktop cooler. Take out a tube only when you want to pipet the sample. Do not remove the tubes from the cooler for more than a few seconds.
4. Vortex the capped tube to mix. Spin the tubes in a tube spinner for a few seconds. Ask your instructor or TA for assistance adding the enzymes. Mix and spin again.
5. Incubate the tubes at 37 °C for 45 - 60 minutes.
6. When the incubation is over, add 2 μL of 6X loading dye to each sample.
Agarose Gel Electrophoresis
1. Load your entire sample into your assigned well in the 0.7% agarose gel made according to the procedure in Lab 7. Record the identity of each well.
- If you are not confident with your DNA loading technique, you can watch a loading technique video here.
- The instructor will load 5-10 μL of the NEB 1 kb Plus DNA Ladder in one lane.
2. Set the voltage to 90 Volts. Run for 90-120 minutes.
3. When electrophoresis is complete, transfer the gel to the ethidium bromide staining solution. Soak for 30 minutes.
4. Next, place the gel in a plastic bag and carry it to the gel imager. Follow imaging instructions, save the image to the computer, and print a copy.
5. Before you leave lab, that your lane info is recorded on the D2L gel image page.
WASTE DISPOSAL AND HAZARDS
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