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Southern blot
Background
• a method to detect the presence of a DNA
sequence in a DNA sample.
• The method is named after its inventor, the
British biologist Edwin Southern.
• Combines agarose gel eletrophoresis for
size separation of DNA with methods to
transfer the size-separated DNA to a filter
membrane for probe hybridization.
• The key to this method is hybridization.
• Hybridization-process of forming a doublestranded DNA molecule between a singlestranded DNA probe and a single-stranded
target DNA.
• There are two important features of
hybridization:
– The reactions are specific-the probes will only
bind to targets with a complementary
sequence.
– The probe can find one molecule of target in a
mixture of millions of related but noncomplementary molecules.
DNA extraction
Procedure
DNA digestion
Agarose electrophoresis
Gel and membrane
preparation
Transfer
Prehybridization
Probe preparation
and hybridization
Wash
Detection
http://www.accessexcellence.org/AB/GG/southblotg.html
DNA extraction
• Quality and amount of DNA is important for southern
blot.
• Agarose electrophoresis check DNA integrity after
extraction.
 High quality intact DNA should give the appearance
of a single band.
 Degraded material will smear downwards.
• Avoid RNA, protein, endogenous nuclease
contamination.
• Avoid DNA degradation.
Enzyme digestion
Restriction endonuclease digestion of
genomic DNA is different from plasmid.
1ug genomic DNA: 5-30U enzyme
Determine appropriate enzyme amount and
digestion time.
Avoid incomplete digestion: use large
volume or use high concentrated enzyme.
Transfer
• Depurination is not required for DNA fragments <10kb
in size. Do not over depurinate, 10mins(or until the
bromophenol blue turns yellow) is usually sufficient for
most samples.
• DNA is then denatured with an alkaline solution such
as NAOH,which causes the double stranded to
become single-stranded.
• DNA is then neutralized with NaCl to prevent rehybridization before adding the probe.
• Transferred by either electrophoresis or capillary
blotting.
• Avoid trapping any air bubbles during transfer.
• Large DNA fragments need overnight to 24h transfer.
Pre-hybridization
• 4-5 hours
• Buffer binds to areas on the blot not
occupied by patient DNA.
• Blocks the empty sites from being bound
during hybridization.
Hybridization and Washing
Lower hybridization temperatures achieve
lower stringency.
65-68℃ is suitable for most long
probes(>100base).
Stringency washes will dependent on the
nature ofn probe and target to be
hybridized. The lower the salt
concentration, the greater the stringency.
The higher the washing temperature, the
greater the stringency.
Watch points
• Using too little DNA-compromise the sensitivity of
the test
• Using too much DNA- poor restriction enzyme
digestion
• Using too high voltage setting for electrophoresisgel to melt or appearance of artifacts
• Improper blocking-high background and
uninterpretable results.
• Insufficient washing-high background and
uninterpretable results.
• Excess washing- dissociate the specific hybrids.