5 replies to this topic

[lyrebird]

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.

[fred_33]

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)?

[lyrebird]

fred_33, on Mar 22 2005, 02:06 AM, said:

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}>

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.

[Lauram]

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

[lyrebird]

Lauram, on Mar 22 2005, 01:34 PM, said:

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.  

[fred_33]

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 1µl of 30µl 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...