Posted 10 January 2005 - 10:36 PM
i am doing northerns but not able see success, i tried with 10ug total RNA and then when no signal detected tried 30 ug RNA now also am not able to see any good signal. i now realised that during transfer before blotting i dint give a wash to the gel to remove formaldehyde, would this be causing anyproblems for transfer or hybridization. however my RNA dot blots are giving really good signals.
thanks in advance
Posted 11 January 2005 - 12:39 AM
Posted 11 January 2005 - 08:55 PM
thanx a lot
Posted 05 March 2005 - 09:16 AM
You could isolate mRNA, but this is a bit costly, and a pain in the ass.
Posted 07 March 2005 - 01:42 AM
first, did you see your RNA on the gel before transfert?
i always rinse the gel before transfert in order to remove formaldehyde and excess of runing buffer salts that are on the gel or above (in ddH2O) for at least 15'. Then i repeat with sterile 10XSSPE.
I put 3Whatman sheets saturated of buffer on the gel and at least 5cm of absorbent towels on top of the whatman and let transfert for 24H.
But i've noticed that if paper sheets and/or membrane are bigger than the gel, transfert is not efficent and buffer goes directly in sheets upper the gel.
Posted 07 March 2005 - 06:26 AM
Yes, I look at the RNA on the gel first. If I can't see a lot of rRNA, then I know I won't see my signal given 1) the size of my probe (.5kb) and 2) the (relatively) low level of expression of my gene.
If your membrane, whatman and towels are so large that they droop over and touch the saturated substrate, then they will wick the buffer up directly without transferring the liquid through the gel.
Also, do you cover the surrounding area with plastic wrap to prevent evapouration? Usually, I cover the whole area with plastic wrap, and then use a blade to cut around the gel/membrane.
I take it that you uv crosslink your RNA before hybridization?
Posted 07 March 2005 - 06:53 AM
i do not cover my preparation. I usually take a big quantity of buffer in order to prevent a lack of buffer in the end of transfer. I did not quantify but i assume i use less than a quarter of the solution.
After the transfert, i dry the membrane by placing it between two sheets of whatman overnight and then fixing the RNA by heating 80° 1h, or directly dry+fix 80° 1h. Then i put the membrane in TBS0.5X/BET for 15', rinse with TBS0.5x for 15' again and then see the RNA by uv light.
The membrane is ok for probing.
Posted 07 March 2005 - 07:42 AM
Could your probe have a low specific activity due to a large amount of starting template? If your dot blots have a lot of nucleic adics on them, then you might get a strong signal for these, even with a low specific activity probe.
Posted 07 March 2005 - 07:47 AM
assuming that in agarose/formaldehyde you're using 30µg of total RNA and that in dot blots 50µg of total RNA are used, the specificity of the probe should not be modified from dot blot to agarose gel...