APPENDIX

Materials Common Chemicals. α32P CTP , BRIT India

α32P dCTP , BRIT India

100bp ladder (G210A) Promega

1kb DNA ladder (G571A)

Acrylamide (75820) USB

Advantage 2 polymerase Mix (639202), BD Clontech

Agarose (75817) USB

Albumin, Bovine pH7 (BSA) (10857) USB

Amino-allyl-UTP-Atto 520 (NU-821-520) Jena Biosciences, GambH

Ammonium Persulphate (76322) USB

Amphicillin (A9518) Sigma

Aphidicolin (A0781) Sigma

Bis Acrylamide (75821) USB

Boric Acid (B6768) Sigma

Bromo phenol Blue (12370) USB

Calf Serum (16170-078) Gibco

CalyculinA (9902) Cell Signaling

Cell culture plastic ware- BD Falcon

Chloroform, Qualigens

Comassie Brilliant Blue G (32812) USB

DABCO ( D-2522) Sigma.

DAPI (D-21490) Molecular Probes

DEPC (D5758), Sigma

DMEM (12430) Invitrogen, GIBCO

DMSO-HybriMax (D2650) Sigma

Ethanol, Merck

Fetal Bovine Serum (16000-044)

Formamide (H5051) Promega

G418 sulfate (11379) USB

Glycine (16407) USB

IPTG (US17886) USB

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Isopropanol, Merck

Kanamycin (K4000) Sigma

Lipofectamine 2000 (11668-027) Invitrogen

Lipofectin Reagent (18292-011) Invitrogen

Lumiphos 530 (Boehringer Mannheim/ Roche)

Luria Agar (M557) Himedia

Luria Broth (M575) Himedia

Media (DMEM, MEM etc.) and TPVG, NCCS Media section.

Methanol, Qualigens

MOPS (19256) USB

Mounting Medium (M1289) Sigma

Nuclease free water (P1195), Promega

Paraformaldehyde (P6148) Sigma

Phosphate Buffered Saline, Dulbecco’s, Ca2+,Mg2+ free (TS 1006) Himedia

Poly-L-Lysine (P4707) Sigma

Precision Plus Protein Standards (161-0373) Biorad

Protease inhibitor cocktail (P8340) Sigma

ProtenaseK (300141), Stratagene

Phenol (75829) USB

RapidHyb Buffer (RPN 1635) USB

RIPA lysis Buffer (sc-24948)

RNA marker (G3191) Promega

SDS (75819) USB

SiRNA Marker (E02450) NEB

Sodium Chloride (21618) USB

Taq DNA polymerase (D-6677) Sigma

Tips (DNase free, RNase free), Axygen.

Tris (75825) USB

Tween 20 (20605) USB

Urea (75826) USB

X-gal (US10077) USB

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Restriction enzymes. BamH1 (R0136L) NEB

BglII (R0144L) NEB

EcoRI (R0101L) NEB

HindIII (R0104L) NEB

Mlu1 (R0198L) NEB

Not1 (R0189L) NEB

SalI (R0138M) NEB

XbaI (R0145L) NEB

XhoI (R0146L) NEB

Antibodies. Ki67 (556003) BD pharmingen

Phospho ATM (Ser 1981) (#4526S), Cell Signaling

Phospho ATR (Ser 428) (#2853S), Cell Signaling

Phospho Histone H2AX (#2577S), Cell Signaling

Phospho pRB (ser-780) (sc-12901-R), Santacruz

Phospho p53 (Ser 15) (#92845), Cell Signaling

Phospho p21 (Thr 145) sc-20220-R, Santacruz

P21 Waf1/Cip1 (#2946), Cell Signaling

P53 (FL-393) (sc-6243), Santacruz

FHIT (#4704), Cell Signaling

actin antibody (clone 14) (#691002), ICN

Anti-GFP antibody (8362), BD Clontech

AlexaFlour 594- goat anti-mouse IgG (A11005), Molecular Probes

AlexaFlour 594- goat anti-rabbit IgG (A11012), Molecular Probes

Cell lines. Hela (ATCC-CCL-2), human cervical cancer

MRC5 (ATCC-CCL-171), Human lung fibroblasts, normal

NIH3T3 (ATCC-CRL-1658) Swiss mouse embryo

Raji (ATCC-CCL-86), human B-cell (Burktt’s) lymphoma

SK-N-MC (ATCC-HTB-10), human neuro-epithelioma

U373 MG, (ECACC-89081403), human glioblastoma, astrocytoma

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WI38 (ATCC-CCL-75), Lung fibroblasts, normal.

CloneM3 (ATCC # CCL 53-1)

Vectors and Plasmids. pTarget Mammalian Expression Vector System (A1410) Promega

pGEM-T easy vector system (A1360) Promega

pGL2 control (E1611) Promega

pGL2 Enhancer (E1621) Promega

pGL2 Promoter (E1631) Promega

pGL2 Basic (E1641) Promega

pRL-CMV control vector (E2261) Promega

pRL-SV40 control vector (E2231) Promega

pSV3 neo β-gal vector (E1081) Promega

pEGFP-N1 vector (6085), BD clontech/Takara

pEGFP-C1 vector (6084), BD clontech/Takara

pLKO.1 puro vector (plasmid 8453), Addgene

Fhit shRNA Constructs (Mouse), HuSH 29mer, Origene Technology

Kits. β-galactosidase enzyme assay system with reporter lysis buffer (E2000), Promega

Access RT-PCR system (A1250), Promega

Bio-Rad Dc Protein estimation Kit, Bio-Rad

ECL Plus Western Blotting Detection system (RPN 2132) GE Healthcare

Epitect Bisulphite modification kit (59104) Qiagen

Great Escape SEAP chemiluminescence kit 2.0 (631737), BD Clontech/Takara

HybSpeed RPA kit (AM1412) Ambion

Luciferase Assay System with reporter lysis buffer (E4030), Promega

QIAfilter Plasmid Midi kit (12243), Qiagen QIAprep Spin Miniprep kit (27106), Qiagen

QIAquick Gel extraction kit (28706), Qiagen

QIAquick Nucleotide removal kit (28304) Qiagen

QIAquick PCR purification kit (28106) Qiagen

Renilla luciferase assay kit (E2810) Promega

Reverse Transcription System (A3500) Promega

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Riboprobe System T7 (P1440) Promega

ShortCut RNAi kit (E2450S) NEB

SuperSignal West Femto Maximum sensitivity substrate (34096), PIERCE

SYBR GREEN PCR Master Mix (4309155) ABI

Taqman Universal PCR Master Mix (434437) ABI

Instrumentation. 3730 DNA analyser (Applied Biosystems, Foster City, CA, USA)

9300 Real time PCR (Applied Biosystems, Foster City, CA, USA)

Cell culture CO2 incubator (Thermo Electron Corp. Vantaa, Finland)

Cell culture Hood (Thermo Electron Corp. Vantaa, Finland)

Confocal Laser Scanning Microscope, (Carl Zeiss, Thornwood, NY)

GeneAmp 9700 Thermal cycler (Applied Biosystems, Foster City, CA, USA)

Forma Orbital Shaker (Thermo Electron Corp. Vantaa, Finland)

Gel Documentation system, G:Box Chemi XL (Syngene, Cambridge, UK)

Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA)

Hybridizer HB-1D, Techne, (Bio-Rad, Hercules, CA, USA)

Florescent Microscope IX81 (Olympus, Tokyo, Japan)

Florescent Microscope BX51 (Olympus, Tokyo, Japan)

Molecular Imager FX (Bio-Rad, Hercules, CA, USA)

Spectrophotometer, SmartSpec 3000 (Bio-Rad, Hercules, CA, USA)

UV cross linker (Stratagene, La Jolla, CA, USA)

Western blotting ,Protean II and Protean III (Bio-Rad, Hercules, CA, USA)

Topcount microplate Scintillation Counter (Packard, US)

Primers. AS89: 5' GGA TAT CCT AAC ACA CCT GAA AAG C 3'

As90: 5' GTT GTA AAC CCC TTC AGC TCC TGC 3'

AS 216: 5’ ACA GCA AGT TTG GAT ATC CTA AG 3’

AS218: 5’ TGT TTT CCT ATG GGG TTG TAA AC 3’

AS217: 5’ CAT CAG AAC CCA GTT CTC CCA TC 3’

AS224: 5’ CCT GGA AGC TCT GTG GGG TCT GGC T 3’

AS164: 5’ CTG CAT CTG TCT TAG GTG CTG G 3’

AS165: 5’ GAT GTC AGG GAT GTG AGA GGT TC 3’

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AS612: 5’ ACC TAA AAT CCC ACA AGA AAG TC 3’

AS613: 5’ CAT TGG GTC CAG GGA ACC T 3’

M3TR taqman 5' 6FAM- CAG ATG TCA GGG ATG TGA -TAMRA 3'

T7 primer 5’ GTA ATA CGA CTC ACT ATA GG 3’

SP6 primer 5’ GAT TTA AGG TGA CAC TAT AGA 3’

M3TR BglIIF: 5’ TCA GAT CTC ACA GCA AGT TTG GAT ATC CTA ACA CAC 3’

M3TR HindIIIR: 5’ TCG AAG CTT CAC TGT TTT CGC TAT GGG GTT G 3’

M3TR-as BglIIF: 5’ TCA GAT CTC CAC TGT TTT CGC TAT GGG GTT G 3’

M3TR-as HindIIIR: 5’ TCG AAG CTT ACA GCA AGT TTG GAT ATC CTA ACA CAC 3’

BglII GFP exp: 5’ CTC AGA TCT GTC GCC ACC ATG 3’

ORF1 Kozak F1: 5' GTC GCC ACC ATG GGG ATG GAA AAA AGA AAA AG 3'

ORF1 HindIII R1: 5' TCG AAG CTT GCA AGC ACA TCT TGG CAT C 3'

ORF2 Kozak F1: 5’ GTC GCC ACC ATG GCA TGG CTG TCC TCT G 3’

ORF2 HindIII R1: 5’ TCG AAG CTT CAC TGT TTT CGC TAT GGG GTT G 3’

ORF3 kozak F1: 5’ GTC GCC ACC ATG CTG GGT GGC CTT TCC TTC AG 3’

ORF3 HindIII R1: 5’ TCG AAG CTT CAG CAA GTT TGG ATA TCC TAA CAC AC 3’

C1: 5' CAC TGG GAC GAC ATG GAG A 3'

C2: 5' CAG GCA GCT CAT AGC TCT TC 3'

C3: 5’ CAG GCA GCT CGT AGC TCT TC 3’

AS436: 5' TGC ACC ACC AAC TGC TTA G 3'

AS437: 5' GGA TGC AGG GAT GAT GTT C 3'

Gapdh Taqman: 5' 6FAM- CAG AAG ACT GTG GAT GGC CCC TC- TAMRA 3'

Fhit F: 5’ CCA GCC TTT TGG AGA TCTGA 3’

Fhit R: 5’ TGG GAA TCG TTT GAG TTA CTC TC 3’

Fhit Met.F 5’ TTT CGG AGA AAA GTA AAG GAA C 3’

Fhit Met R: 5’ AAC AAA AAT ACC GCC TAA ACG 3’

Fhit unmet.F: 5’ TTT TGG AGA AAA GTA AAG GAA T 3’

Fhit unmet.R: 5’ AAC AAA AAT ACC ACC TAA ACA 3’

P53 Met. F: 5’ CGA TCC AAG TGC AGT GTG GAA ATA G 3’

P53 Met. R: 5’ GGT TAT TGC TTT TGC AAA GGA GGA GGT G 3’

P53 Unmet. F: 5’ TGA TTT AAG TGT AGT GTG GAA ATA G 3’

P53 Unmet.R: 5’ CTA CTA TCC TCA ACA TCT TAT TTT TC 3’

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Tissue culture media and reagents.

Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5g/L):

Powdered medium was reconstituted by dissolving in Milli-Q water, The 0.3g/L

glutamine and 3.7g/L sodium bicarbonate was added and pH was adjusted to 7.2-7.4 by

bubbling CO2 gas. The volume was made up to 1 litre, filter sterilized and stored at 4oC.

Minimum Essential Medium, Earle’s (MEM- E): Powdered medium was reconstituted by dissolving in Milli-Q water, The 0.3g/L glutamine

and 2.2g/L sodium bicarbonate was added and pH was adjusted to 7.2-7.4 by bubbling

CO2 gas. The volume was made up to 1 litre, filter sterilized and stored at 4oC.

Opti MEM: Powdered medium was reconstituted by dissolving in Milli-Q water,

The 0.3g/L glutamine and 2.4 g/L sodium bicarbonate was added and pH was adjusted

to 7.2-7.4 by bubbling CO2 gas. The volume was made up to 1 litre, filter sterilized and

stored at 4oC.

Antibiotics stock, P&S (100X): Solutions of penicillin 1X105 units/ml and streptomycin 50mg/ml were prepared in tissue

culture grade water, filter sterilized and stored in aliquots at -20oC till use.

TPVG: 0.1% Trypsin, 0.02% EDTA (versene) and 0.05% glucose in PBS, with phenol red as pH

indicator (1ml of 1% stock). pH was adjusted to 7.4, filter sterilized and stored at 4 oC.

Phosphate buffered saline (PBS): 8.08g NaCl, 0.2g KCl, 1.15g of Na2HPO4, 0.2g of KH2PO4 in 1litre of tissue culture grade

water and sterilized by autoclaving.

G418 sulphate:

50mg/ml stock in tissue culture grade water, filter sterilized, aliquoted and stored in dark

at -200C.

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Common Solutions. EDTA (0.5M, pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 ml of H2O. Stir vigorously on a magnetic

stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into

aliquots and sterilize by autoclaving.

10% SDS: Weigh 10gm of SDS and dissolve by stirring in 90 ml of distilled water. Make up the

volume to 100ml with dist. Water. Store at RT.

Tris-Cl (1M, 8.0): Dissolve 12.11 g of Tris base in 80 ml of H2O. Adjust the pH to 8.0 with conc. HCl. Make

up the volume to 100ml with distilled water. Autoclave to sterilize and store at RT. Other

pH solutions are prepared in similar fashion by adjusting to desired pH.

10X TBE buffer: Dissolve 216 gm of Tris base, 110 gm of Boric acid and 16.6 gm of EDTA in 1.5 liters of

distilled water, by stirring. Make up the volume to 2 liters with dist. Water. Autoclave on

liquid cycle and store at RT.

TE Buffer: Add 1ml of 1M Tris.Cl (pH 8.0, 10 mM) and 50µl of 0.5M EDTA (pH 8.0, 1mM). Make up

volume to 100ml distilled water. Autoclave and Store at RT.

Sodium Acetate: To prepare a 3 M solution: Dissolve 408.3 g of sodium acetate•3H2O in

80 ml of H2O. Adjust the pH to 5.2 with glacial acetic acid. Adjust the volume to 100ml

with H2O. Sterilize by autoclaving immediately. Store at RT.

TEN9 Buffer: Make the final concentration to 0.01 M Tris-Cl (pH 9.0); 0.1 M EDTA (pH 8.0);

0.01 M NaCl, using 1M Tris.Cl pH 9.0, 0.5M EDTA pH 8.0, 5M NaCl respectively.

X-gal: Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a

concentration of 20 mg/ml solution. Use a glass or polypropylene tube. Wrap in

aluminum foil and store at -20°C.

IPTG: Make a 20% (w/v, 0.8 M) solution of IPTG by dissolving 2 g of IPTG in 8 ml of distilled

H2O. Adjust the volume of the solution to 10 ml with H2O and sterilize by passing it

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through a 0.22-μm disposable filter. Dispense the solution into 1-ml aliquots and store

them at -20°C.

10X MOPS: Add 41.8 gm of MOPS in 700ml of DEPC treated water. Adjust the pH to 7.0 with 2N

NaOH. Add 20ml of 1M sodium acetate and 20ml of DEPC treated 0.5M EDTA (pH 8.0).

Makeup the final volume to 1 lit with DEPC treated water. Filter sterilize using 0.45µM

filer. Wrap in aluminum foil and store at 4°C.

20x SSC: For 20x solution: Dissolve 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of

H2O. Adjust pH to 7.0 with Conc. HCL. Autoclave and store at RT. The final

concentrations of the ingredients are 3.0 M NaCl and 0.3 M sodium acetate.

Proteinase K: Weigh the lyophilized powder (commercially available) and dissolve in nuclease free

water at 20 mg/ml concentration.

6x Gel-loading Buffer III: 0.25% (w/v) bromophenol blue

0.25% (w/v) xylene cyanol FF

30% (v/v) glycerol, in H2O.

Ampicillin (100mg/ml): Dissolve 1 gm Ampicillin in 10ml of autoclaved distilled water, filter sterilize, dispense

into 1ml aliquots and store at -20°C.

Kanamycin (30mg/ml): Dissolve 300 mg kanamycin in 10ml of autoclaved distilled water, filter sterilize, dispense

into 1ml aliquots and store at -20°C.

10% APS: Dissolve 1gm of ammonium per sulfate in 10 ml of autoclaved distilled water, dispense

into 1ml aliquots. Protect from light by wrapping in aluminum foil and store at -20°C.

30% Acrylamide: Weigh 29 gm Acrylamide and 1 gm of Bis-acrylamide

Ethidium Bromide: Dissolve 100 mg of EtBr in 10 ml of distilled water. Aliquot, wrap in aluminum foil and

store at 4°C.

SCL solution: Add ProteinaseK to 50 µg/ml final concentration in TE buffer.

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Tampon TFB1: Add 3ml of 1M Potassium acetate, 10ml of 1M KCl, 1ml of 1M CaCl2.2H2O, 5 ml of 1M

MnCl2 and 15 ml of Glycerol. Make the final volume to 100 ml with dist. Water. Filter

sterilizes using 0.45 µ filter. Store at 4°C.

Tampon TFB2: Add 1ml of 1M Na-MOPS pH 7.0, 1ml of 1M KCl, 7.5ml of 1M CaCl2.2H2O and 15 ml of

glycerol. Make the final volume to 100 ml with dist. Water. Filter sterilizes using 0.45 µ

filter. Store at 4°C.

Luria Agar: Weigh 35 gm of LA powder (Himedia, India) in 1 liter of distilled water. Sterilize by

autoclaving.

Luria Broth: Weigh 20 gm of LB pweder (Himedia, India) in 1 liter of distilled water. Sterilize by

autoclaving.

Tris-Glycine-SDS Buffer (5X): Add 15.1 gm of Tris base, 94 gm of Glycine to 900ml of dist. Water. Stir to dissolve. Add

50 ml of 10% SDS and adjust volume to 1 liter with water.

6X SDS loading Dye:

Add 0.5ml of 1M Tris.Cl (pH 6.8), 70µl of β-Mercapto ethanol, 2 ml of 10% SDS and 5 ml

of Glycerol, 0.25% (w/v) bromophenol blue. Make the final volume to 10ml with Dist.

Water. Dispense into 1ml aliquots. Store at 4°C.

1X Phosphate Transfer buffer (for western blot): Add 1.37 gm of Sodium Phosphate Monobasic (Monohydrate) and 2.67 gm of Sodium

Phosphate Dibasic (Heptahydrate) to 1 liter of distilled water.

Fixer solution (for karyotyping): Mix Methanol and Acetic acid in 3:1 ratio. Store at -20°C.

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