My dot blot hybridization doesn't work! - (Sep/06/2007 )

Bacterial growth in these solutions would be bad because bacteria produce proteases, which will break down your antibodies. Milk is a pretty good medium for any number of microorganisms.

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Ooo..Now I know about that. Thanks for the information. On the other hand, I would like to do direct spotting on membrane using non-extracted sample-which is suspended sample. The protocol given is

1. Spot 5 or 10 microlitre of the suspended sample onto the membrane.
2. Let it completely air-dried and then incubate in blotting buffer (7.5x SSC, 4.6M formaldehyde) for 15 minute in 65 degree celcius.
3. After 15 minutes, the buffer was discarded and the membrane was left to air-dried before taken to UV-Crosslink for 5 minute.
4. After UV-crosslinked, the membrane was used for pre-hyb and hyb, next followed by detection and that's all.

I am thinking in the initial protocol there is a step before spotting the suspended sample onto the membrane which is to boil in boiling temperature for 5 minute before spotting. Would you think it is necessary for the boiling and if yes, do the temperature is appropriate?
Thanks for the guidance and help.

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