IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/07/01

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(7/1/14 Gel Electrophoresis)
Current revision (12:41, 15 July 2014) (view source)
(7/1/14 Gel Electrophoresis)
 

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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
Hover name
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
Hover name
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.


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