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Southern Hybridization Strigency Question



Southern Hybridization Strigency Question

A few general questions first:

In Southern hybridization experiments how important is hybridization
temperature? What are the effects of rasing or lowering the hybridization
temperature?


How does one determine the proper washing conditions (assuming 100%
match in base paring).


If you get a weak signal and you know that you have a lot of DNA
on the membrane and that you have good specific activity on your
probe, how do you know if this is because your washing conditions
are too strigent or that the signal is non-specific.


In Current Protocols In Molecular Biology they listed formula for
melting temperature of hybrid nucleic acids:


DNA-DNA hybrid Tm = 81.5 + 16.6(log(M)) + 0.41(%GC) - 0.61(%form)
- 500/N


DNA-RNA hybrid Tm = 79.8 + 18.5(log(M)) + 0.58(%GC) - 11.8(%GC)^2
- 0.56(%form) - 820/L


** where M is molarity of monovalent cations between 0.01M and 0.4M
%GC is percentage of GC pairs
%form is percentage of formamide


My questions are: how does Tm vary when you have monovalent
cations outside of the range 0.01M to 0.4M? The number I get
from the DNA-RNA hybrid Tm formula seems unreasonable. Can anyone
point out corrections?


Does anyone use these formulas for calculating hybridization and
washing conditions? How do you determine your hybridization and
washing conditions?



Now a question specific to my experiment:


I have two identically blotted Southern blots, and I have two 30mer
DNA probes both labelled with 32P to 2*10^8 dpm/ug. Both probes also
have about 70% GC.


I hybridized each probe on a separate membrane under the same
conditions and washed under the same conditions. The first probe
gave me a good signal after a 4 hour exposure on film, and the
2nd probe gave me a very weak signal after 24 hours exposure.


Both probes were pre-hybridized and hybridized in 5xSSC / 5xDenhardt /
1%SDS / (100ug/ml) denatured sheared salmon sperm DNA at 54 degrees.
They were both washed in .2xSSC / .1%SDS at 54 degrees for 1 hour.


Can anyone help me explain my results?
Is my washing condition too strigent for the 2nd probe that I am
washing off my probe? or is my washing condition not strigent
enough that I am getting non-specific hybridizations.


Any theories, comments, references, opinions are welcome. Either
post or e-mail me directly. If I get enough responses I will summarize.
Thanks in advance.
Yng Chen
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I do a lot of hybridizations with ~20 bp probes that are 50% G/C. We
have used a variety of different hyb solutions (reccomended by
manufacturers of different membranes) with little difference observed.
We usually hybridize at 42 degrees C, but 37 gives the same result.
The critical step is washing. I wash first in 2X SSC, 0.1% SDS at 42C
for 10 min, dump out wash (into rad waste) and add more of the same
wash solution for another 15-30 min. Then I scan my membranes with
a hand held geiger counter to check signal/noise. If they are still
way hot, without a noticeable concentration of hot spots, I wash in
1X SSC, 0.1% SDS either at room temp or at 42 depending on just how
hot the filters seem. After 15-30 min of this higher stringency wash
I again check the filters with the monitor. If still too hot, I wash
in 0.5X SSC, 0.1% SDS at room temp for 15-30 min, then put on film.
Longer probes use higher temps for washing (for greater than 500 bp
I use 65 C for hyb and all washing steps).
Stuart Brown
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Last update 12-Nov-2000, Rating of 0 votes.

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