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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
µ
l
20
µ
l
Take 25ml from above
Seperate 75ml
X Assay Buffer
25
µ
l
25
µ
l
Add 0.25 gm Agarose
Eco
R I
3
µ
l
Boil till Agarose dissolves
Hin
d III
3
µ
l
Place Comb (2cm -ve)
Digestion
37o C
(Room Temp)
60 Min
Pour Agarose (60oC)
Gel Loading Buffer
5
µ
l
5
µ
l
Keep Undisturbed
Marker
10
µ
l
Pour 75ml 1x TAE above gel
Control DNA
10
µ
l
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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