genomic southern blotting and dna fingerprinting l.
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0. Genomic Southern Blotting and DNA Fingerprinting. ABE Workshop 2007 Jamie Lum. 0. Southern Blotting is a method to check for the presence of a gene or DNA in a sample. . 0.

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slide3

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1kB WT HM HZ LD 1kb WT HM HZ LD 1kB WT HM HZ LD 1kB WT HM HZ LD

Mark U C U C U C Mark C U C U C U Mark C U C U C U Mark U C U C U C

quantitate dna with serial dilutions

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Quantitate DNA with serial dilutions

In the presence of NBT/BCIP, alkaline phosphatase (AP) precipitates into purple spots.

10-1 10-2 10-3 10-4

Control

Group 1

Group 2

Group 3

Group 4

capillary transfer of the dna from the gel to a nylon membrane
Cut off top right corner of the gel.

Immerse gel in 0.125M HCl and shake for 10 minutes.

Soak the gel in Denaturation Buffer for 30 minutes on shaker.

Rinse the gel with water.

Soak in Neutralization Buffer for 30 minutes.

Cut nylon membrane and two 3M filter papers to the size of the gel. Cut off top right corner of membrane.

Add transfer buffer to a glass tub.

LAYER, LAYER, AND LAYER!!

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Capillary transfer of the DNA fromthe gel to a nylon membrane
slide6

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  • Tray with transfer buffer
  • Wick (3M paper)
  • Gel
  • Nitrocellulose
  • Parafilm cut as a picture frame
  • Filter paper
  • Paper towels
  • Glass dish
  • Weight
  • Allow to sit overnight
slide7

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Mark gel wells on the membrane with a pencil.

Rinse the membrane in 6X SSC for 10 minutes.

Lay the membrane, nucleic acid side up, on a piece of foil and expose to 120,000 microjoules/cm2.

slide8

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Prehybridization and

Hybridization

slide9

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  • Add 10 mL of 42oC DIG Easy Hyb solution to a roller tube with the membrane.
  • Prehybridize for 20-30 minutes in a 42oC hybridization oven.
  • Denature probe in boiling water for 5 minutes and quickly place in ice water.
  • Remove prehybridization solution and add probe/hybridization mixture into roller tube.
  • Hybridize for at least 2 hours in the hybridization oven.
  • Pour off the probe.
slide10

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  • Wash membrane twice in 2X Wash solution for 5 minutes per wash.
  • Wash membrane twice in 0.1X Wash solution for 10 minutes per wash.
  • Equilibrate membrane in Washing Buffer for 2 minutes.
  • Incubate in Blocking solution for 30 minutes.
  • Remove blocking solution and add Antibody solution. Incubate for 30 minutes.
  • Wash membrane twice in Washing Buffer for 15 minutes per wash.
  • Equilibrate membrane in Detection Buffer for 2 minutes.
slide11

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  • Place membrane on a piece of transparency film and spot with CSPD/Detection Buffer over membrane.
  • Wrap up edges, make sure no air bubbles.
  • Tape membrane in pre-warmed X-ray cassette and incubate for 10 minutes.
  • In dark room, place cut X-ray film over membrane and close cassette.
  • Expose the film for 30 minutes.
  • Remove film from cassette and place in developer for 1 minute.
  • Transfer film to fixer for 5 minutes.
  • Rinse with tap water and hang to dry.
group 1

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Group 1

WT HMPDI2 HZPDI2

MW U C U C U C LamHindIII

group 2

0

Group 2

WT HMPDI2 HZPDI2

MW C U C U C U LamHindIII

group 3

0

Group 3

WT HMPDI3 HZPDI3

MW C U C U C U LamHindIII

group 4

0

Group 4

WT HMPDI3 HZPDI3

MW U C U C U C LamHindIII

so how is this information useful

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So how is this information useful?
  • Restriction Fragment Length Polymorphisms or RFLPs differentiates by patterns derived from cleavage of their DNA. The similarity of the patterns generated by RFLPs can be used to differentiate species or individuals from one another.
  • Isolation of DNA for analysis is both time consuming and labor intensive.
  • Polymerase Chain Reaction or PCR is a technique for amplifying a specific region of DNA, defined by a set of two "primers" at which DNA synthesis is initiated by a thermostable DNA polymerase.
  • Used to amplify small amounts of DNA in a few hours so more samples can be analyzed in a shorter time.
  • Paternity testing
  • Positive identification of crime suspect
slide18

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Someone ate the last cookie. A small DNA sample was taken from the plate and DNA was taken from seven of the workshop facilitators. Based on the RFLP, who did it?

1-Dr. Christopher

2-Dong Ping

3-Prof. Kabi

CS-Crime Scene DNA

4-Eun Ju

5-Dr. White

6-Dr. Unabia

7-Dr. Berestecky

1 2 3 CS 4 5 6 7

slide19

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A woman has accused a man of fathering her twins. Of course, he denies it, so a paternity test was done.

1-Mother

2-Child 1

3-Child 2

4-Potential father

1 2 3 4