My horrible Vacuum Blotting Device - (Dec/11/2005 )

Hi everyone,

I just got an vacuum blotter from biorad....my hope was, to get better or even faster
rna-blots....until now, we just did kapilary blotting with good results..but ist takes something
around 4-7 Days to tranfer all RNA....I thought a vacuum blotter might improve that...
but the results are horrible....90 minutes of blotting and most of the rna seemed to stay at the
gel...I just followed the manual...are there any secrets in this technic???

I used:
10 x SSC
Nytra-Membrane (Whatman/Sch&Sch)
Whatman-Paper

transfer at 5 Hg for 90 min

Thanx for your Help

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I would rather use alkaline transfert in my experience that was the best capillary transfert protocol for RNA transfert. It doesn't require so many days I usually transfert overnight and that was enough to transfer even large Viral RNA's
RNA transfert

Hope it helps

Pesji

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Hi,Thanx...

I oncetried the ambion protocol even with that "head over heels" Blotting technic....everybody laught :-)
the rna on the membrane looked ok...but when I tried to hyb it...no signal...what prevents the rna from becomeing completely degraded...( thats what I thought......) ????

K_R

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Ambion is one of the leader companies on RNA manipulation so I think they are pretty serious about how to handle RNA. I've been using succesfully this protocol numbers of time specially to transfer large viral RNA (up to 11 KB). You should just neutralise the blot afterwards like it's described in the protocol

If you use this protocol and didn't get signal that doesn't mean that your transfer didn't work but rather that your hybri^disation was the problem !

Pesji

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