8 replies to this topic

[suhasbn]

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

[pcrman]

After transfer, view you gel under UV to see if most of your RNA has been transferred to the memberane.

[suhasbn]

when i visualize the membrane on the uv i see 80-90% transfer of the RNA from the gel to the membrane. but am not sure if it really stays on it due to formaldehyde even though i fix it with uv in the crosslinker ( if excessive uv causes a problem too for hybridization)
thanx a lot

[Great White Northern]

we never remove our formaldehyde, and have never had problems.  Apparently, it does inhibit adhesion to the blot to a degree, but not very much.  The downside of washing is that your band may not be as clear.  Often, I have problems in that my signal comigrates with the rRNA and masks the true signal (we always get nonspecific hyb to the rRNA due simply to the large amounts).
You could isolate mRNA, but this is a bit costly, and a pain in the ass.

[fred_33]

hi
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.

Fred

[Great White Northern]

hi fred
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?

[fred_33]

hi
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.

Fred

[Great White Northern]

About all I can think of it to use an rRNA probe as a control.  If you have loaded 30micrograms of total RNA, then your cpm should be through the roof.
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.

[fred_33]

hi
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...