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GENOMIC SOUTHERN ANALYSIS

The purpose of the Genomic Southern Analysis is to compare the genomic DNA of wild-type and mutant flies for the presence of restriction fragment length polymorphisms (RFLP). You will use the chromosomal DNA you prepared from mutant flies in lab 3. You will be provided with genomic DNA from the wild-type strain, W1118. You will digest DNA from the common mutant and W1118 with SacII I. You will decide which enzyme to use with your unknown mutants. The digested DNA is then size fractionated by gel electrophoresis and blotted onto a nylon membrane. The process of transferring electrophoretically separated DNA or RNA fragments from agarose gels to nitrocellulose or nylon membranes is known as blotting. This technique of transferring DNA was first described by Edward Southern and the blot became known as a Southern blot. When the technique was applied to RNA, some molecular biologist with a sense of humor dubbed the process Northern blotting; this was followed by Western blots for protein and Southwestern for blots of DNA with attached proteins. Southern and Northern blots are typically used for detecting the presence of a particular fragment or molecule in a specific sample of RNA or DNA. These blots are also used to detect rearrangements in DNA or RNA in a sample. Detection of a particular fragment or of a rearrangement is accomplished by hybridization with a DNA or oligonucleotide probe of known sequence.

You will make two identical blots of the W1118 and the common mutant; one will be hybridized with a your plasmid rescue fragment that has been labeled with biotin. The other blot will be hybridized with a radioactively-labeled probe made from your plasmid rescue fragment DNA. Radioactive signals are detected by placing the hybridized blots on film. Biotin is detected by binding it to strepavidin that has been conjugated with alkaline phosphatase; the phosphatase is used to cause a chemical reaction that can be detected by a color change.

Digestion of chromosomal DNA

You will prepare three Southern Blots for analysis. Two will contain digested chromosomal DNA from W1118 and the common mutant flies; the third will contain digested chromosomal DNA from W1118 and your unknown mutant. If you use different enzymes to digest the DNA from your unknown, you must also digest W1118 DNA with the same enzymes. During Laboratory 8, you will digest the DNA using the general formula below per lane of DNA to be run, use:

 

16 µl chromosomal DNA (subject to change based on the quality of your DNA preparation)
2 µl buffer
2 µl Pst I
20 µl Total       incubate at 37oC overnight

You should know the expected result for the common mutant. What is the size of the fragment of DNA that is expected to give a signal in the common mutant? If more than one signal is detected for this line what are the possible identities of the signals?

The digests should be stopped by the addition of 0.02 M EDTA on the morning following this laboratory.

Genomic Southern Gel

During Laboratory 9 you will size fractionate the digested DNA. Use a large gel box for this. Run a lane of ladder and a lane of W1118 DNA and a lane of mutant DNA. Do this for each individual mutant. Run two sets of lanes for the common mutant and W1118. Skip a lane in the gel between each set of mutants. You are running the different mutants separately with their own lanes of ladder and wild type DNA because each set of digests will be blotted onto separated nylon filters and hybridized with a different probe. Set the gels up as quickly as possible. Run the gel at 120V for 1.5 hours. When the gel is finished running, stain it and cut it to separate the sets for each mutant. Take a picture of each gel fragment. Place a fluorescent rule beside the ladder lane in each and cut a nick in the upper left-hand corner of each gel for orientation purposes.

Preparation of the Gel for Blotting

After you have taken a picture of the gel, you will prepare it for blotting. The DNA will be depurinated by soaking in HCl. The DNA will be denatured and then neutralized. Why are these steps necessary?
  1. Trim the excess from the gel. If you did not do so previously, cut the upper left-hand corner of the gel for orientation.
  2. Place the gel in 0.25 M HCl for approximately 10 min. The time needed may vary with the size and thickness of the gel. In general, the gel should be soaked until the marker dyes change color from a bluish to yellowish color.
  3. Rinse the gel briefly in dH2O to remove the excess HCl.
  4. Soak the gel in a solution of 0.5 M NaOH/0.5 M NaCl for 20-30 minutes.
  5. Soak the gel in a solution of 0.5 M Tris-HCl, pH 7.5l/ 0.5 M NaCl for 20-30 minutes.

Southern Blot

  1. During the last wash prepare the blotting paper, transfer membrane and capillary apparatus. Cut a piece of nylon transfer membrane to the exact size of the gel. Always wear clean, dry gloves when handling the membrane. Cut 6 piece of blotting paper to the exact size of the gel. Cut one longer piece of blotting paper to use as a wick. Half-fill a pan or plastic container with 10X SSC. Place the support for the gel in the center of this. Pre-wet the wick in 10X SSC and place it on the support. Wet 3 of the pieces of blotting paper in 10X SSC and place them on top of the wick. Remove all air bubbles from between the layers of blotting paper using a pipette as a roller.
  2. When the gel washes are finished, place the gel on top of the three pieces of blotting paper. Remove any air bubbles.
  3. Pre-wet the transfer membrane in 10X SSC and place it on top of the gel. Remove any air bubbles.
  4. Wet 3 of the pieces of blotting paper in 10X SSC and place them on top of the membrane.
  5. Line the edge of the blotting paper with plastic wrap to eliminate wicking at any place except directly up through the gel
  6. Stack 4-5 inches of folded paper towels on top of the blotting papers, place a weight on this stack.
  7. Allow the SSC to wick through the membrane and gel overnight. As SSC is drawn through the gel, it will carry the DNA with it. SSC can continue to be drawn through the membrane but the DNA will stick to the membrane.
  8. Disassemble the blotting apparatus. Cut the upper right hand corner of the membrane before lifting it off the gel.
  9. Wash the membrane in 0.4 M NaOH for 1 minute.
  10. Wash the membrane in 0.2 M Tris-HCl, pH 7. /1X SSC for 1 minute.
  11. UV crosslink the DNA to the membrane.

 

Northern Blot

A Northern Blot is exactly the same as a Southern Blot, except that there is no need to denature or neutralize the RNA in the gel before blotting. It is already denatured. It is a good idea to remove some of the formaldehyde, however, since it may interfere with transfer. After you have taken a picture of your RNA gel.(Don't forget the fluorescent ruler), wash the gel 3 times for 10 minute each in dH2O. Then proceed to set up a blot exactly as described above. You may also skip the NaOH and Tris-HCl, pH 7. /1X SSC washes after the gel has soaked overnight.