LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
sample was reduced to 10 μl or less. Later the filter containing the sample was inverted
into a new tube and centrifuged at 10000 rpm/ 3 min/ RT. Volume of the sample
collected was measured and made up to 10 μl.
3.9.2 Preparation of cDNA:
Total RNA from test as well as control samples was treated with DNase
wherever required as described in section 4.3.3 and later subjected to cDNA synthesis
as described below.
Reaction mixture-1
RNA (2.5 µg/µl) 10 µl
Oligo dT (Invitrogen; 1 µg/µl) 6 µl
Random Primer (Invitrogen; 0.5 µg/µl) 2 µl
A mixture of Oligo dT and random primers was made and 8 μl was dispensed on each
tube containing RNA. The tubes were incubated at 65 °C for 10 min and immediately
transferred on ice and incubated for 10 min.
Reaction mixture-2
5X FS Buffer 6 µl
0.1M DTT 3 µl
50X aadUTP.dNTP mix 0.6 µl
RNase IN 0.5 µl
Superscript SSII 1.5 µl
D/W 0.4 µl
12 μl of this reaction mixture was added to each tube and was incubated at 42 °C for 1
h in water bath. The reaction was terminated by adding 10 μl 0.5M EDTA. 10 μl of
freshly prepared 1 N NaOH was added to degrade RNA the reaction mixture was
incubated at 65 °C for 10 min and cooled at RT.
3.9.3 Purification of cDNA
To the newly synthesized cDNA 25 μl 1 M HEPES buffer pH 7.0 was added.
Reaction volume was made 500 μl with nuclease free water and contents were
transferred to microcon column. Samples were centrifuged at 10000 rpm/ 10 min/ RT.
Flow through was discarded and again 450 μl nuclease free water was added to the
column. Samples were again centrifuged at 10000 rpm/ 10 min/ RT and continued to be
centrifuged for small intervals so as to reduce the volume