Transfer Membranes

Protocol 11: Oligonucleotide Probes to be Used in Northern and Southern Blotting
For Pure Nylon Neutral Membranes
Non Isotopic Detection Systems
1. Pre-Hybridization Conditions:
5X SSPE
1.0 % SDS
50 % Formamide
5X Denhardt's Reagent
Pre-Hybridization be performed at TH (Hybridization Temperature), for 1 – 2 hours.
NOTE: High concentrations of blocking reagent such as Denhardt's will quench signal. Concentrations of up to 10X
can be used. Some common substitutions for Denhardt's Reagent are Heparin, Gelatin, and commercially produced
blocking powders. Non-fat dry milk is not recommended for use due to it's potential for quenching signal.
2. Remove the Pre-Hybridization Buffer
3. Hybridization Conditions:
5X SSPE
1.0 % SDS
50 % Formamide
20 µg/ml oligonucleotide
Hybridization should be performed in a shaker bath for 1 hour at TH.
4. Washing Conditions: Wash the blot twice for 5 minutes each in 500 ml 2X SSPE and 0.1% SDS at room
temperature.
5. Final Wash Conditions: Wash the blot twice for five minutes each in 500 ml of 0.1X SSPE at TH.
Isotopic Detection Systems
1. Pre-Hybridization Conditions:
6X SSPE
0.5 % SDS
5X Denhardt's Reagent
Pre-Hybridization should be carried out at TH for 30 minutes to 1 hour
2. Remove the Pre-hybridization Buffer
3. Hybridization Conditions:
6X SSPE
0.5 % SDS
5X Denhardt's Reagent
100 – 200 µg/ml tRNA
Hybridization should be carried out with 10 6 – 10 7 cpm/ml end labeled probe at TH.
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 TH.
11
protocol
o c o l s
© 2008 AppliChem • Transfer Membranes 33