Transfer Membranes

protocol
10
Protocol 10: RNA Probes to be Used in Northern and Southern Blotting
RNA Probes with Pure Nylon Neutral Membranes
1. Pre-Hybridization Conditions:
5X SSPE
1.0 % SDS
50 % Formamide
10X Denhardt's Reagent
Pre-Hybridization be performed for 1 – 2 hours between 50 – 60°C.
NOTE: For probe denaturation, incubate the probe at 100°C for 5 minutes, then immediately chill on ice.
2. Remove the Pre-Hybridization Buffer
3. Hybridization Conditions:
5x SSPE
1.0 % SDS
50 % Formamide
20 ng/ml RNA Probe
Hybridization should be performed in a shaker bath for 1 hour at TH.
4. Washing Conditions: Wash the blot three times in 1X SSPE and 0.1% SDS for 20 minutes each at 65° C.
5. Final Wash Conditions: Wash the blot in 0.1X SSPE and 0.1% SDS for 20 minutes at 65° C.
RNA Probes with Pure Nitrocellulose Unsupported and Reprobe Nitrocellulose
Supported Membranes
1. Pre-Hybridization Conditions:
6X SSPE
0.5 % SDS
5X Denhardt's Reagent
Pre-Hybridization should be performed at 60°C for 30 minutes to 1 hour.
2. Remove the Hybridization Buffer
3. Hybridization Conditions:
6X SSPE
0.5 % SDS
5X Denhardt's Reagent
100 µg/ml RNA
Hybridization should be carried out with 10 6 – 10 7 cpm/ml end labeled probe at TH for up to 20 hours.
4. Washing Conditions: Wash the blot three times in 1X SSPE and 0.1 % SDS for 20 minutes at 65°C.
5. Final Wash Conditions: Wash the blot in 0.1X SSPE and 0.1 % SDS for 20 minutes at 65°C.
NOTE: Background removal is accomplished by incubating the blot in 1.0 µg/ml RNase A in 2X SSC for 20 minutes at room
temperature. Next rinse the blot in 1X SSPE and 0.1 % SDS.
p r o t
32 Transfer Membranes • AppliChem © 2008