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.
| Uncut Plasmid Check
|
Reagent
|
Volume
|
Expected:
1. TesA = 2721
2. mCherry = size
3. TetR = 2124
4. Lac = 2125
5. RBS= 2063
|
15 μL/lane; 1% agarose; Ladder
|
|
DNA(plasmid) |
5.0 μL
|
|
10X buffer |
2.0
|
|
dH2O |
13.0
|
|
|
20.0 μL --> 37°C/ ~40 min.
|
| Insert Size Check
|
Reagent
|
Volume
|
Expected:
All Backbones = 2070 1. TesA = 651
2. mCherry = size
3. TetR = 54
4. Lac = 55
5. RBS= 13
|
15 μL/lane; 1% agarose; Ladder
|
|
DNA(plasmid) |
5.0 μL
|
|
10X buffer |
2.0
|
|
EcoRI |
1.0
|
|
PstI |
1.0
|
|
dH2O |
11.0
|
|
|
20.0 μL --> 37°C/ ~40 min.
|
|
Project name
Main project page
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