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min. with
a
gentle shaking followed by soaking in 250 ml of denaturation solution (I
M NaCI, 0.5
M
NaOH) in a glass tray with gentle shaking for 90 min. on a platform
shaker and the gel was washed 4 times with distilled water. Then, the gel w;is soaked
in 250 ml of neutralization solution (1.5
M
NaCI, 0.5
M
Tris HCI, pH 7.0) with
shaking for 9 0 min. on a shaker. After 90 min., the gcl was rinsed twice with 250 ml
of sterile distilled water.
A
paper wick was kept on a platform in such
n
way th;tt the
ends were dipped well into 20X SSC (175.5 g NaCI. 80.4 g trisodium citrate and 1000
ml distilled water, pH 7.0) Three Whatman No.3 sheets, cut to the size of the template
were wetted with 20X SSC and arranged over the paper wick. The gel was placed on
this in such a way that the bottom side of the gel was facing upwards. A nylon
membrane (Hybond, Boehringer and Mannheim,
UK)
cut to the size of the gel, was
first wetted in sterile distilled water, then soaked in 2X SSC and placed over the gel.
Three Whatman No.3 sheets cut to the size of the gel were moistened with 20X SSC
and placed over the nylon membrane and one layer of dry Whatman No.3 sheet was
kept on the top. Further stacking was done to a height of 1.5-2.0 inch with paper
towels. The blot was removed after an overnight transfer and rinsed briefly in
2X
SSC
for 30 s, air-dried and the DNA was immobilized onto the membrane (Sambrook
et
al., 1989).
3.2.3.5.3.2.5.3.1. Preparation of probe DNA
Approximately 25-30 ng of probe DNA
(cab
or
Chi
or
Ltp)
was used for
radioactive labeling. Radiolabeling of probe DNA was performed by random primer
0lig0 labeling method using Random Primer Labeling kit (Bangalore Genei Pvt. Ltd.,
Bangalore, India). A 35 pl reaction mixture contained 25-30 ng probe DNA, 1.5 p1
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