Genomic DNA cutting - (Jan/19/2007 )
when you digest genomic DNA you get a smear. If you lane is properly loaded (not to much DNA, not to little) you sometimes see bands which usually correspond rRNA genes. These gene are repeated so many times in large block that they can form noticible bands.
Now, if your smear is too dark, it could mean the lane is over loaded... it is hard to give a description of a nicely loaded lane. As lane widith can very, and the sourse of genomic DNA, (genome size, thus copy number of the fragment you are looking for) can vary too.
Now if you have a smear, worry not. (the centre of the smear should be around 3kb). Only worry if the smear has a peak intensitty around 500bp. If the peak centres around 500bp, you DNA has been degraded, which mean that your prep was not clean enough, which in turn resulted in your DNA degrading during the digest.
As mdfenko has alluded, you need to southernblot your DNA. So you need too transfer your DNA to a nitrocellulose sheet, the cheapest way is to use capilary action helped along with paper towels. YOu will also need a marker of some sort, etc radioactive marker if you are using a radioactive probe.
Look up Southern blot for details ;- google.
Once you have your DNA on a nitrocellulose sheet, denature the DNA, vaccume dry it, fix the DNA by UV . etc etc
Then you hybridise your nitrocellulose sheet with a probe that is specific to the DNA you are looking for.
This is another series of protocols... all depending the type of probe you are using, eg floresence, radioactive
Fine tuning probing conditions so there is a strong signal without nonspecific binding.
Develop the image. And you should see your DNA lit up.
But is this what you want?
What do you want to do?
thank you very much for your reply. i will do what u said and may be i will contact after i get result. thank you very much once again.
okay. Just read up the protocol for southerblot and the probing method you want to use.
It is very long and can easily be messed up.
And remember to add a marker!! That will show up on your final image