Plant Biology And Plant Biotechnology
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Preparation of DNA Preparation of Agarose Gel
Component Vial 1 Vial 2 Vial 3 Vial 4 Prepare 100 ml od 1x TAE
Lambda DNA 20 µl 20 µl Take 25ml from above Seperate 75ml
X Assay Buffer 25 µl 25 µl Add 0.25 gm Agarose
EcoR I  3 µl Boil till Agarose dissolves
Hind III  3 µl Place Comb (2cm -ve)
Digestion 37o C
(Room Temp)
60 Min
Pour Agarose (60oC)
Gel Loading Buffer  5 µl  5 µl Keep Undisturbed
Marker 10 µl Pour 75ml 1x TAE above gel
Control DNA 10 µl Gently lift comb without disturbing wells
Electrophoresis
Connect Power cord to Electrophoretic power supply
(Red = Anode; Black = Cathode)
Load samples in the well in desired order
Set desired voltage and switch on power
When the tracking dye (Bromophenol Blue) from the well reaches 3/4th distance of the Gel switch off power.
(Approx 60 minutes)
STAINING PROCEDURE TO VISUALIZE DNA
Prepare 1X Dye

Carefully transfer gel from tank to tray containing Dye
Make sure that Gel is completely immersed in Dye
Place on rocker (or Intermittent shaking) -Uniforn staining
Pour out stain and destain with Distilled water
DNA visible as DARK bands in Light Blue Background



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