| HUMID CHAMBER. Layer the sides of a rectangular tupperware dish or large petri dish with wet paper towels. Place parallel strips of tygon tubing or glass rods on the bottom of the dish (to serve as a platform for the slides). Cover the container with the lid to seal the humid chamber. | DETECTION OF DIGOXIGENIN LABELLED PROBES ANTIBODIES: 1) Mouse anti-digoxigenin IgG1 (monoclonal) obtained from Boeheringer Manheim Biochemicals (catalog # 1333 062). 2) Goat anti-mouse IgG (H+L) fluorescein (FITC) conjugated (affinity purified) obtained from Jackson Immunoresearch Laboratories catalog # 115-095-003 (phone 1-800-367-5296). and 3) Swine anti-goat IgG fluorescein conjugated (affinity purified) obtained from Boeheringer Manheim Biochemicals (catalog # 605270). Just prior to use, all three antibodies are diluted 1 to 250 using 10% horse serum |
| 10% HORSE SERUM: 100% horse serum (heat inactivated) was obtained from Gibco BRL (catalog # 230-6050AG) and filter sterilized. The working blocking reagent is 10% horse serum in 1X TBS (with 0.02% sodium azide). The formula for one liter of 10X TBS is; 80g NaCl, 2g KCl, 30g Tris base dissolved in 800 ml H20 then pH adjusted to 7.4 using 1M HCl. Bring to 1 liter using H20 and autoclave | DETECTION OF BIOTINYLATED DNA PROBES Use Chromosome In Situ Kit; Signal Amplification Reagent Set (Catalog # S1352-SET) from ONCOR, 209 Perry Parkway, Box 870, Gaithersburg, Maryland 20877 |
| PREPARATION of DNA for MAKING PROBES 1) Digest CsCl purified cosmids with Sau3A 2) Phenol/CHCl3 extract 1X, then CHCl3 extract 1X 3) EtOH ppt (add 1/10 vol 5M NaCl, 2 vol 100% EtOH) 4) Resuspend in 1X TE pH 8.0 |
DIGOXIGENIN LABELLED DNA PROBES. The enzymes for nick translation were obtained from the BioNickTM Labelling System from GIBCO BRL (Catalog # 8247SA). The protocol is similar to that described for biotinylation except that 1 ul Digoxigenin DNA labelling mixture (Boehringer Mannheim Biochemicals; catalog # 1277 065) and 5ul 10 X nick translation buffer (0.5 M Tris-HCl pH 7.8, 50 mM MgCl, 100 mM beta-mercaptoethanol, 100ug/ml BSA) are substituted for 5ul 10 X dNTP mix from the BioNickTM Labelling kit. Note: The BMB Digoxigenin DNA labelling mixture contains all the required nucleotides including digoxigenin-UTP. It is listed as 10X but I use it as a 50X solution for nick translation. |
BIOTIN LABELLED DNA PROBES. Use BioNickTM Labelling System from GIBCO BRL (Catalog # 8247SA)
a) Set up 3 tubes of Sau3A digested DNA; tube A 300 ng DNA, tube B 100 ng DNA and tube C 30 ng DNA
b) Add reagents from BioNickTM kit to each tube as described in kit
NOTE: we use less DNA than the kit suggests but add reagent volumes as stated for 50 ul reaction using 1ug DNA c) Incubate for 1 hr at 16℃
d) Add 5 ul 10X stop buffer
e) Precipitate DNA by adding 5 ul 10 mg/ml sonicated salmon sperm DNA, 6 ul 3M Sodium Acetate and 120 ul cold 100% EtOH.
f) Incubate 2hr at -20℃.
g) Pellet DNA by microfuging 15 minutes and discard supernatent
h) Resupend in 100 ul H20.
i) Reprecipitate DNA by adding 10 ul 3M Sodium Acetate and 200 ul cold 100% EtOH. Incubate 2hr at -20℃.
j) Pellet DNA by microfuging 15 minutes and discard supernatent.
k) Wash pellet with 1 ml cold (-20℃) 70% EtOH. Vacuum dry pellet.
l) Resuspend DNA in 25 ul 1X TE pH 8
