Northern hybridization - plz help (Jun/09/2006 )

hi all,


could some one please tell me that waht should be the minimum socn of the RNA on blot for the good hybridia\satin.



how can i c use the ladder for checking the size of my gene on the blot. i want to ask how can compare the size of my gene of intest.



waht shd be the minimum cocn of the labeled ATP for the northern hybridisatin.

>could some one please tell me that waht should be the minimum socn of the RNA on blot for the good hybridia\satin.
For radioactive method, I see lots of protocols suggesting a minimal of 10 ug of total RNA for Northern analysis.

>how can i c use the ladder for checking the size of my gene on the blot. i want to ask how can compare the size of my gene >of intest.
To determine the size of your gene of interest on the blot, plot a MW (nucleotide) vs. relative-migrating-distance graph. This is done by either referring to an agarose gel photo (most probably) or the acquired X-ray film (if you're using rna radioactive ladder). Then determine the size of your gene by refering to its relative-migrating-distance on the X-ray film to acquire its MW. This is done by referring from the graph you've plotted.

>waht shd be the minimum cocn of the labeled ATP for the northern hybridisatin.
You should refer to the labelling system you're using. The manual provides is a good guidance.

minimum is roughly 15µg for gel analysis and 50µg for dot blots.
i use fresh ATP 6000Ci/mmol but some protocols do good with 3000Ci/mmol