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Southern problem - No signal


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5 replies to this topic

#1 lyrebird

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Posted 21 March 2005 - 03:40 PM

I am trying to test the knockout result by southern blotting.
I used radioactive labeling, positive charged nylon membrane and carefully followed the classical protocol. I was quite sure that the transferring was successful. But after exposure, I could see nothing in the film. ;) I repeated twice but nothing changed. Would anyone like to tell me any possible reasons for this result and what I should do? Any comments are highly appreciated.

#2 fred_33

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Posted 22 March 2005 - 01:06 AM

hi
after transfert, did you colored with ethidium bromide membrane and gel (for see the transfert on the membrane and what remains on the gel)?

#3 lyrebird

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Posted 22 March 2005 - 11:55 AM

hi
after transfert, did you colored with ethidium bromide membrane and gel (for see the transfert on the membrane and what remains on the gel)?

<{POST_SNAPBACK}>


:P I only did that to the membrane and saw clearly the DNA smear. Does that mean the transferring was ok? I didn't check what left on the gel.
Many thanks for your reply. :)

#4 Lauram

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Posted 22 March 2005 - 12:34 PM

What's the amount of the DNA that you load on the gel? Maybe you need more! I'm using 10microgram per well.

#5 lyrebird

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Posted 22 March 2005 - 02:14 PM

What's the amount of the DNA that you load on the gel? Maybe you need more! I'm using 10microgram per well.

<{POST_SNAPBACK}>

Thank you. But I tried approximately the same amount of sample as you did. :P

#6 fred_33

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Posted 23 March 2005 - 01:57 AM

hi
a dna smear signify that transfert was ok. to check the efficiency better i colour the gel too.
Did you quantify the labeling of the probe?
did you run on pAcryl gel a part of your probe (i run 1Ál of 30Ál labelled probe)?

did you purify your probe after labelling?
i am ondering if your hybridization and or washing conditions are not too stringent for your probe...




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