DIG-Northerns

jon at molbiol.net jon at molbiol.net
Tue Sep 17 06:27:32 EST 2002


There are many sources of peppered spots with DIG-Northerns -

(i) Precipiate in probe - solution: filter (can not filter if protein or milk based blocker)
(ii) Agarose flecks or SSC crystals on membrane - solution: wash membrane post-blot with 2*SSC for 10 minutes
(iii) Antibody precipitate - solution: spin 10 mins full speed at 4 degrees Celsius
(iv) Probe aggregate - solution: filter (possible only in EasyHyb or other SDS-based hyb solutions)
(v) Alkaline phosphatase contamination - presumably the ubiquitous alkaline phosphatase is present on dust from human skin and bugs in the lab, keeping everything shut must help avoid this
(vi) Check solutions for precipitate and make fresh regularly
(v) Mg in detection buffer - it isn't necessary, and MgCl2 precipitates readily on the bench so leave it out

There isn't an easy answer - I still get spots, but not always too bad.  One example of a real nice one I did is here:

http://www.r9corporation.fsnet.co.uk/Methods/dignorthernf.htm

Read all the literature, and go from there...

Hope this helped...

Jon

rspathis at binghamton.edu wrote:

> I am having trouble with background on my Northern Blots.  The first few times I
> used the system it worked great, lately though my films havew been peppered with
> tiny spots.  As I am trying to quantitate the bands this has become quite a
> problem.  I think that I have tried everything.  Changing membranes, spinning
> down the Anti-DIG antibody, being careful not to let the blot dry out....any
> ideas??

> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&begin=0





http://biowww.net/mynews/tree.php?group_name=bionet.molbio.methds-reagnts&begin=0



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