Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 141

stored for a long time at room temperature (RT). Turbid, contaminated media
must be discarded.

4. LB agar: Add 15 g agar (Sigma, St. Louis, MO) to LB, autoclave, and allow

medium to cool to 50 ° C before adding antibiotics and pouring plates. Plates can

be stored for 1 mo at 4 ° C. Dry the plates in 37 ° C incubator overnight before use.

5. Nylon membrane: for example, Hybond N+ (Amersham, Arlington Hts., IL).

6. Whatman 3MM papers.

7. Denaturation solution: 1.5 M NaCl, 0.5 M NaOH (can be stored at RT).

8. Neutralization solution: 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.2 (can be stored at RT).

9. 1 M NaPi solution: 684 mL 1 M Na 2 HPO 4 , 316 mL 1 M NaH 2 PO 4 , pH 7.2 (can be

stored at RT).

10. L

sheared salmon sperm DNA, 10 g bovine serum albumin (BSA). Fill to 1 L with
deionized water (can be stored at RT).

Church buffer: 500 mL 1 M NaPi, 330 mL 20% SDS, 1 mL 0.5 M EDTA, 10 µ

11. Random priming labeling kit for DNA. For example, Rediprime (Amersham).

12.

32 P-CTP (Amersham). The half-life of 32 P is approximately 14.3 d. Care should
be taken when handling radioactive isotopes. Refer to local safety rules.

13. 20X SSC: 175.3 g NaCl, 88.2 g Na-citrate, adjust pH to 7.0, autoclave (can be

stored at RT).

14. Wash solution 1: 2X SSC, 1% SDS (can be stored at RT).

15. Wash solution 2: 0.4X SSC, 1% SDS (can be stored at RT).

2.2. Generation of Constructs
2.2.1. Definition of the Probe
1.

Restriction enzymes and buffers (store at -20C).

2. T7 sequencing kit (Pharmacia, Uppsala, Sweden).

2.2.2. Mutations Using the Selection Cassette and Cre/loxP System
1. Neomycin resistance cassettes: pMC1 Neo poly A vector (Stratagene, LaJolla,

CA) containing a promoter, a neomycin resistance gene, and a polyadenylation
signal; pNeo plasmid (Pharmacia) containing only the neomycin resistance gene;
pPNT with a strong PGK promoter driving the neomycin resistance gene (24) ;
neo-tk cassette flanked by loxP sites (Fässler, R., unpublished).

2. Restriction enzymes and buffers.

3. T4 DNA ligase and ligation buffer (Boehringer Mannheim, Mannheim, Germany).

2.3. Isolation of Feeder Cells
1.

Mice expressing a neomycin (or hygromycin or puromycin) resistance gene.

2. C57BL6 female mice.

3. 70% Ethanol.

4. Sterile dissecting equipment.

5. 10X phosphate-buffered saline (10X PBS): 80.06 g NaCl, 2.01 g KCl, 14.42 g

Na 2 HPO 4 , 2.04 g KH 2 PO 4 . Fill to 1 L with DDW and autoclave (store at RT).