Northern transfer - how to make sure complete and efficient transfer has taken place (Feb/21/2009 )
I was just wondering when you blot a gel onto a nitrocellulose membrane, how to make sure that complete and efficient transfer has taken place. I am running a northern blot. If after the transfer, I don't see any fluorescence under UV in the gel, does that mean that complete transfer took place? Also I was wondering how important it is to place parafilm on the edges of the gel during the transfer. I tried it but without any success. Can anyone show me how to go about doing it? I know these are very basic questions but I am learning things. Hope I can get some kind of answers.
Thanks in advance.
In general, yes, you are correct. But, if the transfer is to long the nucleic acids can go throw the membrane and then you lose them.
It is very important to place paraphilm on the edges. It forces all of the buffer to go throw the gel and the membrane. You can also use saran rap instead and gust cut out the square for the gel.
When first tested methods like transfer times we always used multiple membranes. You can stack a few and load some samples to test optimal transfer conditions before using your actual samples.
Parafilm (or plastic wrap) is very important during transfer or the capillary action will not be forced through the membrane and you will get inefficient transfer. Also, don;t forget to x-link to your membrane! Nitrocellulose is not activated like Hybond+ so x-link is needed.
The other common step for checking transfer is to stain the membrane with methylene blue. Look for the clear transfer of ribosomal bands (assuming it's total RNA) and no bubbles.
Thank you all for your suggestions!
I was wondering about x-link. I am using nitrocellulose in my lab.
Another query regarding hybridization, does the hybridization buffer make a difference? if i m using riboprobes, the hyb buffer composition should be different from that used for dna probes. Is that important? i guess as far as my understanding goes, if you probe with riboprobes, you need to work with more stringent conditions so the prehyb and the hyb temp is 65 and not 40. am i rite? so what is the reason? is it because the riboprobes bind unspecifically? but if the probe is very specific to my gene of interest, then why am i working with such stringent condition? i think i m also getting cross hybridization to ribosomal rna. the post doc in my lab wants me to do a very stringent washing like skip the mild washing and go straight to the 0.1X SSC, 0.1% SDS wash 3 times for 20 mins at 65 degree. But the problem is I am not getting good signals even when exposed for 3 days.
in my lab we use the same hub buffer for dna and rna probes but i saw a protocol where they recommend different hyb sol composition......why is that?
can any one explain me in simple way to know the exact amount/activity of probe to see a good signal? I know it depends on the quantity of gene of my interest present..this is how i prepare the riboprobe (Maxiscript kit)
10X buffer 2 ul
10 mM ATP 1 ul
10 mM GTP 1ul
10 mM CTP 1 ul
32P-UTP 2.5 ul
H20 1 ul
DNA template 9.5 ul
T7 polymerase 2 ul
After incubation I dilute it with 50 ul RNase free water and then column purify it. So how much probe should I add to the hyb solution?? the post doc just told me to not dilute the probe too much. So I put 80 ul of above probe to 10 ml of hyb solution.