xzhang at riker.neoucom.edu (Xiaolan Zhang) wrote:
>>Subject: Bands in all lanes [SOUTHERN]
>Organization: Northeastern Ohio Universities College of Medicine
>>Hello fellow bionetters,
>>I am performing a genomic southern using 6 REs and DNA from 2 species. My
>probe is a 70bp fragment from the 5' end of my cDNA which I have labeled
>by random priming. The probe does not appear to contain repeat sequences.
>The DNA was digested well and transferred to nylon membranes.
>Hybridization at high stringency and exposure of X-ray film for 1 week was
>used. The blots are very clean.
>>My results have been consistently puzzling. There are 8 bands ranging in
>size from 1 to 8 kb that appear in all lanes regardless of RE or specie.
>These bands vary in intensity but are about the same as the specific
>pattern over which they are superimposed.
>>I think that if there was a contaminating DNA in the samples it would not
>yield the same bands after digestion with different REs. I tested my
>loading buffer for contamination by running just buffer and found none.
>There should not be so many introns covered by such a short probe. And
>they could not all happen to have the same RE sites.
>>What could these bands be from and how can I solve this puzzle?
>>My E-mail address: xzhang at riker.neoucom.edu>
I cannot explain for your observation. but as i know random labelling
require at least 200bp to work. So my suggestion is either increase the
length of your template or use 32P end labelling to do your Southern to
see whether the result will improve.
Email: h8913564 at hkucc.hku.hk