7/1/14 Gel Electrophoresis
- Gel Electrophoresis of Plasmids
- Made TAE buffer with 1 L of distilled water and 20mL of 50X stock of TAE. Also prepared the gel with agarose and water mixture and let solidify.
- Made plasmids ready by using 3μL of DNA and 3μL of loading dye. 5 tubes of this were made as well as 1 tube of ladder 3μL.
- Insert ladder as well as DNA plasmids in wells and let sit for around 45-50 minutes.
- This was to determine concentration of plasmids
- Next, the sizes of the plasmids were tested to see how accurate they are compared to the literature value
- Used the plasmids and cut them with restriction digests
- 2μL of DNA, 1μL of Buffer, 5μL of water, 2μL of, 1μL of E and 1μ of P restriction digests.
- Heated in a 37°C water bath for around 40 minutes
- Plasmids that have been cut were taken out and loading dye was input--2μL
- This along with the ladder was filled into the wells and the machine was run for around 35 minutes. The voltage was increased from 80 V to 100 V
- Pictures were taken of both gels and data recorded.
| Reagent
|
Volume
|
Expected:
1. BB 1 = size
2. BB2 = size
|
15 μL/lane; 1% agarose; Ladder
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| EcoRI |
1.0
|
| PstI |
1.0
|
| dH2O |
9.5
|
| |
15 μL --> 37°C/ ~40 min.
|
|
Project name
