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Southern Blot hybridization

Southern Blot

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#1 studentT

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Posted 02 August 2012 - 12:07 PM

Instead of boiling my probe to denature in labeling solution I accidentally added it to Express Hyb, then realized my mistake and boiled the whole 30ml of hybridization buffer + probe for 20 minutes in 95C heating block. Do you think the SDS and whatever else that might be in Express Hyb is going to interfere with probe denaturation at 95C?

Thanks

#2 bob1

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Posted 02 August 2012 - 01:09 PM

Probably not I would guess. However, you will probably be better off boiling a new aliquot of the probe and re-probing, as you will have to do this anyway to confirm that what you may or may not see is the correct result.

#3 studentT

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Posted 02 August 2012 - 01:39 PM

Thanks for the reply. Since this is a confirmation SB of samples already screened by PCR and expected band sizes are known, I will just sit and wait for the film to expose. I am not sure if there is anything chemical at all that might prevent dsDNA from denaturing at 95C.

#4 uday

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Posted 24 September 2012 - 05:39 PM

I did the same mistake last week (add the labelled probe without heating/boiling for denaturation) but realized later on. So I continued developing the phospho-image and surprisingly expected bands were shown up with minimal background probing. Later on I reprobe the same blot to confirm it again. I am also wondering how I got the bands first time even though denaturation is a critical step for hybridization. Any comments ??
I am just curious to know whether you got any bands ?





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