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NORTHERNBLOT


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5 replies to this topic

#1 A_Gisela

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Posted 02 July 2009 - 01:43 PM

Hi everybody!!

I am having a very stupid problem but i cant get a logical cause. It is the first time i set up a northernblot and somehow is not working at all. I am working with RNA from S. coelicolor M145. The method i am using is northernblot by capilarity because on my lab we dont have any machine to blot. My specific problem is that i can not transfer the RNA from the agorose gel to the membrane. I already tried to let the blot longer, with more buffer, with more weight, but nothing is working. The RNA agarose gel is made with formaldehide. The buffer i use to blot is the SSC 10X (NaCl 1.5 M, and Sodium Citrate 0.15 M). Do you have any idea about what is happening?, Do you know if i have to make any other treatment to the RNA, or to the RNA agarose gel before blotting?......PLEASEE HELP!!!!....I will be really glad for your answer. <_<

#2 bob1

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Posted 02 July 2009 - 04:26 PM

Make sure that the filter paper or your stack is the same size as your gel, if it is larger, it will flop over the edges and suck up buffer straight from th tank/bridge rather than drawing it through the gel. This is known as short circuiting and can also be prevented by covering the edges of the bridge in parafilm.

Have a look at the attached image - blue is the buffer tank, pink is the bridge and black is the parafilm.

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  • blotsetup.jpg


#3 A_Gisela

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Posted 02 July 2009 - 06:06 PM

Thanks a lot for your answer, at the beggining we though that was the problem but we already tried that, so that is not the reason :( . Any other idea?

#4 eldon

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Posted 03 July 2009 - 05:58 AM

Thanks a lot for your answer, at the beggining we though that was the problem but we already tried that, so that is not the reason :( . Any other idea?


Did you stain the gel after transfer to see if anything is there?

Try downward passive transfer....far superior for southern or northern...faster, more efficient, uses gravity, won't flatten your gel etc.

check out ambion for protocol and figs etc.

http://www.ambion.co.../tn/73/733.html

#5 A_Gisela

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Posted 03 July 2009 - 07:09 AM

Hi eldon!

Yes i did, I checked the gel and all my samples were still there. Dont know what is going on, the RNA doesnt want to go to thye membrane, it is just staying on the agarose gel...... :(

#6 bob1

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Posted 06 July 2009 - 04:18 PM

Try using 20x SSC, the higher salt content should increase the mobility of the RNA.




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