STUDIES ON A NOVEL NON-CODING RNA...
Transcript of STUDIES ON A NOVEL NON-CODING RNA...
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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