Telomeric Repeat Amplification Protocol

The reagents for telomeric repeat amplification protocol (TRAP) are as follows:

Material/Reagent

Supplier

Cat. no.

2-Propanol (Isopropanol)

Sigma

I-9516

40% Polyacrylamide/bisacrylamide

Bio-Rad

161-0144EDU

stock (19/1)

Ammonium persulfate

Bio-Rad

161-0700EDU

Bio-Rad Protein Assay Kit

Bio-Rad

500-0002

Boric acid

Sigma

B-0252

Bromophenol blue

Sigma

B-6131

EDTA

Sigma

E-5134

EGTA

Sigma

E-3839

Ethanol

Filter paper

Fuji Medical X-ray Film (Super RX)

03G050

Glacial acetic acid

Glycerol

Sigma

G-2025

Heat block

Magnesium chloride (MgCl26H2O)

Sigma

M-2670

Nonidet P-40 (NP40)

Sigma

N-6507

PAGE vertical gel apparatus

Phosphorlmager™

Phenylmethyl sulfonyl fluoride

Sigma

P-7626

Potassium chloride (KCl)

Sigma

P-4504

Potassium phosphate (KH2PO4)

Sigma

P-5655

Power supply

Pronase

Calbiochem

537088

Ribonuclease (RNase) inhibitor

Gibco-BRL

15518-012

Sodium acetate

Sigma

S-8625

Sodium chloride (NaCl)

Sigma

S-3014

Sodium deoxycholate

Sigma

D-6750

Sodium phosphate (Na2HPO47H2O)

Sigma

S-9390

Sterile H2O

SYBR® Green

Molecular Probes

S-7563

T4 polynucleotide kinase

Gibco-BRL

18004-010

Taq polymerase

Gibco-BRL

10342020

TEMED

Bio-Rad

161-0800EDU

Thermocycler (PCR)

TRAPeze® Telomerase Detection Kit

Intergen Company

S7700

Tris base

Sigma

T-1503

Xylene cyanol

Sigma

X-4126

ß -mercaptoethanol

Sigma

M-7522

32P-ATP

PerkinElmer

BLU502H

EDTA, ethylenediaminetetraacetic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid.

EDTA, ethylenediaminetetraacetic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid.

2.1.1. TRAP Solutions 1. NP40 Lysis buffer: a. 100 mL Stock buffer

10 mM Tris-HCl, pH 7.5 1 mM MgCl2 1 mM EGTA 150 mM NaCl 10% glycerol Sterile H2O

1 mL of 1.0 M stock 1 mL of 0.1 M stock 1 mL of 0.1 M stock 10 mL of 1.5 M stock 10 mL 77 mL 100 mL

b. Before use, make up the working solution as follows: 4 |L of 0.1 M phenylmethyl sulfonyl fluoride (17.4 mg/mL in 2-propanol), 1.4 |L ß-mer-captoethanol, 0.84 |L of 0.12 M sodium deoxycholate, 10 |L NP-40, 40 |L RNase inhibitor (10 U/|L). Bring to 4 mL with NP40 stock buffer. 10X Phosphate-buffered saline (PBS): 80 g NaCl, 2 g KCl, 11.5 g Na2HPO47H2O, 2 g KH2PO4, pH 7.3, to 1 L with sterile H2O. 10% Polyacrylamide stock in 400 mL of 0.5X TBE: 100 mL of 40% polyacrlamide solution (19:1), 40 mL of 5X TBE buffer, 260 mL deionized H2O. Final vol 400 mL. 50 mL of 10% Polyacrylamide gel: 49.5 mL of 10% polyacrylamide stock (19:1), 0.5 mL of 10% ammonium persulfate, 0.05 mL TEMED. Final vol 50 mL. 50X TAE buffer: 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL of 0.5 M EDTA, pH 8.0. Add sterile H2O to 1 L and autoclave.

5X TBE buffer: 54 g Tris base, 27.5 g boric acid, 20 mL of 0.5 M EDTA. Add deionized water to 1 L.

TRAP 1 L Gel fixative: 29.2 g NaCl, 3.28 g Naacetate, 800 mL 50% ethanol; pH 4.2, to 1 L with 50% ethanol.

5 mL Gel loading dye solution: 2.5 mL of glycerol, 1.0 mL of 1.25% bromophenol blue, 1.0 mL of 1.25% Xylene cyanol, 0.5 mL of 0.5 M EDTA, pH 8.0. Final vol 5.0 mL.

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