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Hybridization with Random Primed Probes 1. Prepare Hyb solution. Plan for 0.2 ml/cm2. For mini-blot, try 10 ml. For large blot, try 20 ml.
 

Summary: 46
Hybridization with Random Primed Probes
1. Prepare Hyb solution. Plan for 0.2 ml/cm2. For mini-blot, try 10 ml. For large blot, try 20 ml.
Use tightly sealed plastic tray. Combine ingredients in the order listed.
Southern Northern
30% or 50% 6 ml Formamide 10 ml
5X SSC 5 ml 20X SSC 5 ml
ddH2O 6.3 ml ddH2O 3.1 ml
2.5X Denhardt's 1 ml 50X Denhardt's 1 ml
25 mM KPi, pH 7.4 0.5 ml 1M KPi, pH 7.4 0.5 ml
0.5% or 0.1% SDS 1.0 ml 10% SDS 0.2 ml
0.1 mg/ml ssDNA 0.2 ml ssDNA 0.2 ml
20 mls 20 mls
2. Float Southern or Northern blot in 5X SSC. Submerge to wet completely. For glyoxal gels, make
sure to incubate membrane in 25 mM Tris, pH 8 for 5 minutes at 65C prior to this step.
3. Place blot in HYB solution in sealable tray or bag. Prehybridize for 1 hour at 37C.
4. Random primed probe: Boil probe for 5 minutes to denature. Use tube cap lock. Chill on ice.
Remove 1 ml of HYB solution from tray, mix with denatured probe, add back to tray. Use the
following amount of probe: 1 X 106 cpm/ml (1 - 10 ng/ml or 0.1 - 0.2 pmol/ml).
5. Incubate overnight at 37C. HYB temperature should be < Tm-25 (see below).

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine