Screening for Mutations in Cartilage ECM Genes - Methods in Molecular Biology: Extracellular Matrix Protocols - page 128

4. 1 Kb and 100 bp DNA ladder (Gibco-BRL).
5. 6% polyacrylamide solution. For 500 mL: 100 mL of 30% w/v acrylamide solu-
tion (37.5:1 acrylamide: bis —Protogel™), 100 mL of 5X TBE solution, 300 mL
of dH 2 O.
6. 5X TBE solution. For 5 L: 270 g Tris, 138 g Boric Acid (AnalaR-BDH), 100 mL
0.5 M EDTA (Gibco-BRL).

7. SSCP—Silver stain solutions.

a. Gel fixative solution: 10% ethanol, 0.5% acetic acid. 50 mL 100% v/v etha-
nol, 2.5 mL acetic acid glacial

100% (AnalaR-BDH) in 500 mL of dH 2 O.
b. Gel staining solution: 0.15% silver nitrate. 0.5 g silver nitrate (Sigma) in 300
of mL dH 2 O.
c. Developing solution: 2.25 g NaOH (AnalaR-BDH), 0.6 mL formaldehyde
(Sigma) in 150 mL of dH 2 O.
d. Stop solution: 0.75% sodium carbonate. 0.75 g sodium carbonate (AnalaR-
BDH) in 100 mL d H 2 O.

≈

8. Gel drying kit (Promega, Madison, WI).

2.4. Cloning of PCR Products
1.

LB Broth. 20 g bacto-tryptone (Gibco-BRL), 10 g bacto-yeast extract (Gibco-
BRL), 20 g NaCl (AnalaR-BDH) make up to 2 L with dH 2 O, adjusted to pH 7.0
and sterilize by autoclaving.

2. For LB agar, add 15 g of agar (Gibco-BRL) to 1 L of LB broth and sterilize

by autoclaving.

3. LB agar plates containing carbenicillin (Sigma) (50 mg/mL). Approx 1 h prior to

transformation spread each plate with 50 mL of X-Gal (BIO-RAD) (40 mg/mL in
dimethylformamide [Sigma]).

4. Original TA Cloning © Kit (Invitrogen, San Diego, CA).

5. QIAquick PCR Purification Kit and QIAprep Spin Miniprep Kit (Qiagen).

2.5. DNA Sequence Analysis of Cloned PCR Products
1. ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit

(Perkin-Elmer Applied Biosystems, Norwalk, CT).

2. M13 Forward (-20) primer (5'-g taa aac gac ggc gac-3') and M13 Reverse (5'-cag

gaa aca gct atg ac-3') primer.

3. Sequencing reaction buffer: 450 m M Tris-HCl, pH 8.0 (Gibco-BRL), 10 m M

MgCl 2 (AnalaR-BDH).

3. Methods
3.1. Isolation of genomic DNA and RNA
3.1.1 Isolation of RNA
The procedure described below is suitable for the isolation of total RNA
from one 25 cm 2 flask of confluent cells ( see Note 1 ).