Download Southern_Hybridization2

Southern Hybridization
Lab
SH
• SH – done to know whether a specific DNA
sequence is present in a DNA sample and
where it is located with respect to restriction
enzyme sites
• Restriction digestion + Electrophoresis +
Hybridization = contains specific DNA
sequence or not
Steps
• Digestion + Gel
• Agarose gel is soaked in a basic solution
– to denature DNA fragments
• Gel – neutralization buffer
• Gel is placed on a long piece of blotting
paper with ends of paper suspended in a
reservoir of salt solution
• Nitrocellulose or nylon membrane is laid
on top of gel
Steps
• Blotting paper and a stack of dry absorbent paper (such
as paper towels) are then placed on the membrane
• Capillary action – fluid is drawn from reservoir up
through gel and into stack of dry paper.
- Carries denatured DNA fragments up with it out of gel
onto membrane (stick forming same pattern) only
- Denatured DNA fixed to it through heating or exposure to
ultra-violet
- Membrane with fixed single stranded DNA –
hybridization with desired probe
Steps
• Prehybridization buffer – prevents probe from binding to
the membrane in a nonspecific manner
• Hybridization buffer – contains labeled probe (molecule
called biotin)
- Solution contains two-component molecule (protein
conjugate)
- One component is streptavidin which binds tightly to
biotin
- Other component is enzyme alkaline phosphatase
- Both bind to biotin labeled probe.
Steps
• After hybridization – membrane is rinsed
repeatedly under conditions that will
remove unhybridized and
nonspecifically bound probe – this will
not disrupt hydrogen bonds between
probe and target sequence in sample
DNA
Color development solution
• 5-bromo-4-chloro-3 indolyphosphate (BCIP) and
nitro tetrazolium (NBT)
• Alkaline phosphatase portion of conjugate
removes a phosphate group from BCIP –
resulting product dimerizes to form a dark
blue ppt
• Dimerization step also releases hydride ions
that reduce NBT – purple ppt
• Precipitate is present where ever probe is bound
to the membrane – indicating location of DNA
fragments hybridized to probe
Preparation and experiment
schedules
• April 22 – preparation of solutions
• April 29 – Steps A, B, C and D
• April 30 – Steps E, F, and G
• May 1 – Step H
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This project is funded by a grant awarded under the President’s Community Based Job Training Grant as
implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
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against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in
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President’s Community-Based Job Training Grants as implemented
by the U.S. Department of Labor’s Employment and Training
Administration. The solution was created by the grantee and does
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