LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
2. 25 mM MgCl 2
3. 10 mM dNTPs
4. 10 pM forward primer
5. 10 pM reverse primer
6. Taq. polymerase
7. Filter sterilized water
8. DNA sample dissolved in TE (template)
9. Thermal cycler (Tetrad2-Peltier thermal cycler from Bio-Rad)
Method:
PCR reactions were carried out in sterile thin walled 0.6 ml capacity
eppendorf tubes. PCR reactions were carried out in 25 μl reaction volumes containing
100 ng of DNA template, 10 µM of each forward and reverse gene specific primers
(Table 2), 500 µM each of dNTPs, 2 U of Taq polymerase and 1.5-2.5 mM MgCl2 and
PCR buffer containing 10 mM Tris HCl pH 8.3 and 50 mM KCl. Reaction mixture
without DNA served as negative control. Thermocycling was performed using DNA
thermal cycler. PCR products were visualised on 1.6% agaorose gel as described in
section 8.2.
3.4.4 Activity of ATM gene:
For one sample activity of ATM gene was compared between cell line and
PBL by immunostaining for pATM.
Materials:
1. Cobalt-60 isotope source (Bhabhatron-II)
2. 0.1% lysine (Sigma) coated cover slips
3. 4% paraformaldehyde (PFA)
4. 0.3% TritonX-100 in PBS
5. BSA
6. Anti pATM antibody
7. Anti mouse Alexa-546 secondary antibody
8. DABCO (1,4-diazabicyclo[2.2.2]octane) (4% w/v DABCO in 80% v/v glycerol in
PBS)
Method:
0.3x10 6 cells from both the cell line and respective isolated lymphocytes were
seeded in 1ml complete medium and irradiated with 4 Gy radiation dose using a
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