I'm using DIG Northern starter kit by Roche. I'm trying for weeks and following the protocol (transfer in SSC 20x over night, but i tried also the trasfer in SSC 10x, the membrane is a Nylon positively charged one) but the morning after i still find the higher MW RNA (over 2,5Kb) in the gel. What should i do?
I use this type of blotter (http://e-taitec.com/products/products-detail-en.php?machine_name=G%20capillary%20blotter%20D%20set) with 20x SSC. In my case, transfer is completed in 4 hours. How do you set up your transfer?
I use this setup (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/articles/biology/southern-and-northern-blotting/southern-northern-blot-transfer-assembly.jpg). The only difference is that between the Whatman bridge and the gel I put 3 wet Whatman 3MM papers .
I guess you need to look a little backwards and see what gel are you running? Agarose or polyacrylamide (PA) ? concentrations? These are important questions.. Passive transfer from Agarose is more efficient as compared to PA gels due to low porosity of PA gels? In such case, either increase the transfer period or use an electroblot apparatus..
I use 5x SSC + 10 mM NaOH. Capillary transfer for large RNA (9 kb RNA marker) is complete in 3 h. If you have some RNA to spare, you can run that in one lane and after transfer, cut off that piece of the membrane and stain the RNA using methylene blue to confirm transfer.
Waqas i use 1% agarose gel, at first i loaded 1ug of total mRNA per lane, then i started loading 2-5ug.
Matthew I tried to go on with the protocol and I had trouble during the detection too (the background was really strong and the signal both of the probe of the gene that I'm studying and of the beta-actin was too weak). Anyway thanks for your advises. Maybe I will try by reducing the agarose concentration and extending the transfer time.
Philip I used the methylene blue to confirm transfer but i'm afraid that even if I see that the rRNA is present on the membrane, my gene of interest - that has a higher MW - could be left on the gel. In fact, when I analyze the gel the next morning I can see that the marker for the highest MW are still present on the gel, while the lowest are absent. Moreover the protocol of the kit advise against using alkali transfer, hence I think that I can't use the transfer buffer that you're using. Anyway thank you for your advise.
Hi Tiziano, I would say go for 0.6-0.7% Agarose and do extend transfer time.. If you have enough of RNA sample, may be you should run identical samples in few different lanes and do transfer to membrane for different time lengths. And see what you get. Please do let us know of the outcome, excited to see your results. Another important factor, as for detection of your target, do you know how abundant your target mRNA is, as this varies depending upon various factors, e.g. the promoter under whom control it is.. You may try to use a higher concentration like ~10 ug for mRNA targets which are not abundant.. thanks
I tried with a 1% agarose gel but this time I made a thinner gel. I tried probing with the beta-actin probe O/N but today I still had trouble with the detection -a very strong background with a very low signal-. Since I'm using an old Nitrocellulose membrane it can be possible that it has lost its positive charge?
Tiziano Siri Hello maybe you know if the Nitrocellulose membrane really lost it's positive charge and is it possible? I have the same problem - a very strong background with a very low and not good signal. I did Northern blot about a year ago and I got good signals even with the smaller amount of total RNAs and now I can't repeat it.
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