Bands in all lanes [SOUTHERN]
Harry.Witchel at bristol.ac.uk
Tue Nov 7 14:26:32 EST 1995
In article <DHMGE5.8LK at hkuxb.hku.hk>, username at host.hku.hk says...
>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
Aha! No one can suggest anything, so I will suggest something that has
already been excluded: contamination, particularly plasmid contamination. The
typical test for plasmid contamination is to run one lane of your southern
without any digestion whatsoever -- if you still get the bands, then you have
plasmid contamination. Because plasmids are small, they often are lacking the
REs you may be using. Furthermore, a single plasmid will have several bands
associated with it: supercoiled, nicked circular, etc.. If you have multiple
plasmids of different sizes (a glassware problem), then your enzymes can even
digest your plasmids and you will still get multiple bands.
If you do have plasmid contamination, you will have to throw out all
your solutions for making DNA, clean your pipetmen with some product such as
DNA away, and regularly clean your glassware with 1:10 HCl soak overnight.
The safest way to be certain is to use all plastic disposible ware, but this
is not so easy for high speed ethanol precipitations. The first paragraph on
genomic DNA preparation in The Red Book (Ausubel -- "Current Protocols in
Molecular Biology") gives a scary warning as to the number of man-hours wasted
with contaminated DNA. If you do have contamination, it is better to find out
now, rather than to go to the trouble of cloning and sequencing everything
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