MCB 730Dr. Showalter

SOUTHERN BLOT LAB: BACKGROUND, OVERVIEW & PROTOCOL

Background

In 1975, E. M. Southern developed a technique to transfer DNA directly from an agarose gel to a nitrocellulose or nylon membrane. The technique relies on capillary action. The DNA, once bound on the membrane, can then be used for hybridization analysis to determine the presence and location of a particular DNA sequence. In the past decade, Southern's original paper on the technique was the most frequently cited paper in research.

Restriction digestion, electrophoresis and staining allow us to cut DNA molecules into reproducible fragments and to look at the sizes of those fragments. However, it does not give much information about the DNA base sequence within the fragments. Often it is important to know whether a specific DNA base sequence is present in a sample and where it is located with respect to restriction sites.The technique most commonly used to address these issues is hybridization analysis.

Hybridization analysis involves separating (denaturing) the strands of the DNA molecules to be analyzed and mixing those strands with a labeled, single-stranded DNA or RNA probe. If the probe's sequence is complementary to a DNA molecule, then hybridization (or annealing) can occur under the appropriate conditions. The sample can be rinsed to remove excess probe (and non-specific hybridization) and tested for the presence of hybridized probe. Hybridized probe indicates the presence of the DNA sequence of interest.

One Southern blothybridization application is the screening of DNA libraries. For example, a genomic DNA library is a collection of clones that, taken together, represent the entire genome of an organism. To find a plasmid containing a specific DNA sequence (or gene of interest), the entire library is transformed into a bacterial host. The transformants are plated onto agar to produce individual colonies, each representing one fragment of the organism's DNA, "lifted" onto a membrane, and the DNA denatured and probed with the sequence of interest. If colonies are identified, they can be transferred to separate cultures, and plasmid DNA prepared for further analysis and use.

Another major application of Southernblot hybridization analysis is the testing of the products of a restriction digest to determine which fragments, if any, contain a certain DNA sequence, or to determine the size of a fragment containing that sequence. The procedure most commonly used for this is called "Southern gel blotting"and is followed by hybridization analysis.

Day / Gel hybridization / Colony Hybridization
April 4
(Monday) / Genomic DNA isolation
PCR amplification(TA) / Genomic DNA isolation
PCR amplification(TA)
April 6
(Wednesday) / Electrophoresis & transfer / Ligation and transformation
April 7
(Thursday) / Membrane cross linking and membrane stored at 4°C / Plates stored at 4ºC
April 11
(Monday) / - / Colony lifting
April 29
(Friday) / Probe labeling and overnight incubation at 37°C / Probe labeling and overnight incubation at 37°C
April 30
(Saturday) / Probe stored at -20ºC (TA) / Probe stored at -20ºC (TA)
May 2
(Monday) / Prehybridization, hybridization and overnight incubation at hybridization temperature / Prehybridization,hybridization and overnight incubation at hybridization temperature
May 4
(Wednesday) / Washes and detection / Washes and detection

Southern Blot Lab: Overview

Materials needed:

0.2 N HClDIG High Prime labeling and detection kit

0.5 M NaOH/1.5 M NaCl20X, 2X SSC

1.5 M NaCl/0.5 M Tris-HCl, pH 7.40.2M EDTA (pH 8.0)

Nitrocellulose membranes

Paper towelsWhatman 3MM filter paper

Glass plateSponge

Pyrex dishWeight

Tupperware dish/or sealable bagShaking incubator or water bath, temperature controlled

Southern Blot Lab: Detailed Protocol

  1. Genomic DNA extraction and PCR amplification

Genomic DNA was extracted using the CTAB method and the sequence of interest was amplified using PCR.

PCR set up:

2 l Genomic DNA (25-100ng/l)

2.0l 10X buffer

0.25 l dNTPs

0.25 l Taq DNA polymerase

1 l Forward primer

1 l Reverse primer

Add water to 20l total volume

Genomic DNA and PCR product along with the markerwas run on a mini-gel for1.5- 2h.

If you want to hybridize restriction digested genomic DNA, you need:

5 g genomic DNA

2 l 10X buffer

1 l enzyme (10 U/l)

Add water to 20 l total volume

Digest overnight at 37°C andrun on a mini-gel for 1.5-2 h.

  1. Gel
  2. Placegel, in 0.2 N HCl for 10 min, then rinse with water
  3. Place in 0.5 M NaOH/1.5 M NaCl(Denaturation solution). Incubate twice for 15min.
  4. Place gel in 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 (Neutralization solution). Incubate for 30 min and rinse with water
  5. Assemble overnight blot (semi-dry blot).
  6. Take apart the blot the next day and UV cross-link(or bake for 30 min at 80oC) to bind the DNA to the membrane.

C. Setting up the gel for transferto nitrocellulose(i.e., blotting)

(A) Sponge method transfer set up:

Place a sponge, slightly larger than the gel, in a glass tray and fill with enough 20X SSC so that the soaked sponge is about half submerged. When the sponge is fully saturated, layer 3 pieces of Whatman 3MM paper. Wet the filter paper thoroughly and remove all air bubbles. Drain the gel and lay on top of the filter paper being careful to avoid air bubbles. (Clip off one corner of your nitrocellulose and record which corner you removed. This will help you orient your blot to the picture of your gel.) Lay the nitrocellulose exactly on top of the gel. Be sure the nitrocellulose does not hang over the gel (it should fit exactly on top) and remove any trapped air bubbles. Now add, in order, 3 more pieces of Whatman filter paper, 2 inches of paper towels, a glass plate and top it off with a small weight. Cover the edges of the tray with plastic wrap. Allow the transfer to proceedovernight.

  1. Preparation of the target
  2. Colony lift
  3. Carefully lay the membrane onto the pre-cooled agar plate. Beginning at one edge of the plate lay the membrane down smoothly, avoiding bubbles.
  4. Mark the membrane and plate with a distinctive pattern
  5. Remove the membrane from the plate after 1 min. A second lift may be performed if necessary. Seal the master plate with parafilm and store at 4oC until needed.
  6. Place the membrane, colony-side up, onto filter paper saturated with 0.5 M NaOH/1.5 M NaCl (Denaturation solution). Incubate 5 min.
  7. Briefly blot the membrane on dry filter paper.
  8. Place the membrane, colony-side up, onto filter paper saturated with 1.5 M NaCl/0.5 M Tris-HCl, pH 7.4 (Neutralization solution). Incubate for 5 min.
  9. Briefly blot on dry filter paper.
  10. Place the membrane, colony-side up, onto filter paper saturated with 2X SSC for 5 min.
  11. UV cross link DNA (or bake for 30 min at 80oC) to bind the DNA to the membrane.
  1. Make the probe.
  2. Add 1g of the template DNA and sterile, double distilled water to a final volume of 24l.
  3. Denature the DNA by heating in a boiling water bath for 10 min and quickly chilling in an ice/water bath.
  4. Mix DIG-High prime (vial 1) thoroughly and add 6l to the denatured DNA, mix and centrifuge briefly
  5. Incubate 1 h or overnight at 37oC
  6. Stop the reaction by adding 2 l 0.2M EDTA (pH 8.0) or heating to 65oC for 10 min

Day 1

A. Hybridization

Hybridization analysis is a little like fishing (for DNA). If you know a little about the DNA sequence you're interested in, locating it shouldn't be too difficult. Using that sequence, you can design a probe (the "hook") and the nitrocellulose-bound DNA provides the pond. The rest is just methodology and it is the same for a gel or colony lift.

Prehybridization and hybridization of the membrane

  1. Preheat an appropriate volume (10ml/100 cm2 filter) of DIG Easy Hyb to the appropriate hybridization temperature (to be announced, 45°C).
  2. Place the nitrocellulose membrane in a sealable bag or plastic container (Tupperware). Add enough prehybridization solution (i) to fully cover the membrane and prehybridize 30 min, shaking gently, at the hybridization temperature.
  3. Denature DIG-labeled probe by boiling for 5 min and rapidly cooling in an ice/water bath.
  4. Add15l denatured DIG-labeled probe (iii) to the preheated DIG Easy Hyb (i) and mix well (but gently to avoid bubbles that increase background).
  5. Pour off the prehybridization solution (ii) and add probe/hybridization mixture (v) to the membrane
  6. Incubate 4 h to overnight at the hybridization temperature with gentle agitation.

[The appropriate hybridization temperature is calculated according to GC content andpercent homology of probe to target according to the following equation:

Tm = 49.82 + 0.41 (% G + C) - (600/l) [l = length of hybrid in base pairs]

Topt. = Tm – 20 to 25°C]

Day 2

Washes and detection

A. Stringency washes

Rinse the filter as follows:

  1. 2 X 5 min in 2X SSC, 0.1% SDS at RT. with constant agitation
  2. 2 X 15 min in 0.5X SSC, 0.1% SDS (pre-warmed to wash temperature) at 65oC

B. Detection

  1. Rinse the membrane briefly (1-5 min) in Washing Buffer
  2. Incubate for 30 min in 100 ml Blockingsolution.
  3. Incubate for 30 min in 20 ml Antibodysolution
  4. Wash 2 X 15 min in 100 ml Washing buffer
  5. Equilibrate 2-5 min in 20 ml Detection buffer.
  6. Incubate the membrane in 10 ml freshly prepared color substrate solution in the dark. Do not shake during color development. Monitor the color development (may take up to 16 h).
  7. Stop the reaction, when the desired spot or band intensities are achieved, by washing the membrane for 5 min with 50 ml sterile double distilled water or TE buffer.
  8. Results can be documented by photocopying the wet membrane or by photography.

Important Notes:

Genomic DNA was extracted from Arabidopsis wild type plants (Lab 2, Dr. Wyatt lab). The gene we are interested is a vesicle-associated membrane protein, a member ofSynaptobrevin-like AtVAMP7C, v-SNARE protein family (At5g11150). The full length gene is of 1.8 kb and the coding region is 1.2kb The PCR product of 1.2kb coding region was cloned into the TOPO vector (Lab 3, Dr. Wyatt’s lab). As a confirmatory step, the agarose gel with the genomic DNA and the PCR product along with the marker after electrophoresis will be transferred onto the nitrocellulose membrane and a Southern hybridization will be done. The PCR product cloned into the TOPO vector will be used as a probe for the experiment. A signal in the genomic DNA lane confirms the presence of the gene. The PCR product was run as control for the experiment.

For the colony lift, the transformed E. coli cells from Dr. Wyatt’s lab (Lab 3) will be used. The bacteria were plated on selective media that contain X-Gal for blue/white screening. The cells contain the TOPO vector into which a part of the right border T-DNA was cloned. The same probe will be used for the colony lift and the conventional blot. The students have to identify clones that contain the insert versus cells that carry the re-ligated vector.

The protocol for this lab was adapted from Roche DIG High Prime DNA labeling and Detection Starter Kit 1 (Cat. No. 1 745 832 910).

Conclusions:

Did the probe recognize the genomic DNA (or colonies) from the gel? If so, what does that mean? If not, why not?

Lab Report (100 points, due at the beginning of lab onMay 11, 2011):

Your lab report should include the following sections and describe everything that was done in the style of a PNAS paper. Each student must prepare and submit their own lab report.

  • Abstract
  • Introduction (on the method and objectives)
  • Results
  • Discussion
  • Materials and methods (summarized protocol)
  • Acknowledgements
  • References
  • Answers to the following Questions:
  1. Who invented Northern blotting and Western blotting?
  2. What is a Southwestern blot?
  3. Explain another way to label a DNA probe for Southern hybridization analysis?
  4. What is Southern dot blot analysis and what is it used for?
  5. What two hybridization parameters or conditions are typically altered for maximum hybridization?
  6. What is a zoo blot and a garden blot?
  7. What kinds of nucleic acid bind to nitrocellulose? What kinds of nucleic acid do not bind?
  8. Why do you soak the gel and not the colony lift in 0.2N HCl?
  9. Explain how the DIG-labeling and detection kit that you used works.

10.When doing a northern blot, why do you avoid soaking the gel in 0.5 M NaOH/1.5 M NaCl as you do in Southern blotting?

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