Title of lesson: Probing for Genes

Source/Author(s): Restriction digest, Southern blotting, and hybridization protocols modified from Biology 4106: Molecular Genetics Laboratory Manual, Baylor University, Spring 1997, compiled by Dr. Christopher Kearney. Gel electrophoresis protocol modified from Access Excellence Activities Exchange, online at http://www.accessexcellence.org/AE/AEPC/geneconn/castgel/

Target Audience: High school Biology I (honors) or Biology II

Lesson plan:

I. Performance or learner outcomes

The student will be able to:

Use the scientific method to perform an experiment, practice safe laboratory techniques, cut DNA with restriction enzymes, perform agarose gel electrophoresis, perform Southern blotting, perform DNA hybridization record an accurate laboratory observations, and write a reflective lab report that interprets the findings of their experiment.      

II. Overview:  This investigation involves the Human Genome Project’s goal of learning about other organisms to better understand ourselves, and will last for two and a half weeks.  The students will identify commonalities between mice, pigs, and humans, then select a gene they might share.  They will then perform an experiment in which they will probe pre-extracted mouse, pig, and human DNA for this human gene (the human DNA serves as a control).  Techniques include using restriction enzymes to cut DNA, agarose gel electrophoresis, and Southern blotting.  Students will also keep a lab journal reflecting their daily participation in the experiment, and they will write a lab report documenting and interpreting their work.  This lab investigation requires much out-of-class preparation by the teacher to fit the experiment into a 50-minute bell schedule.  If it is possible for the students to set aside one day as an in-shool “field trip”, they will be able to complete more of the steps themselves.

III. Resources, materials and supplies needed: 

Equipment: Computers for internet access, waterbath, micropipettors (p20), gel electrophoresis kits (agarose minigel) that include chambers, lids, pouring trays, combs, and power supply, refrigerator, freezer, microwave, hybridization oven, vacuum drying oven, shaker, and camera (digital or Polaroid).

Supplies:  Spiral for lab journal, non-powdered latex gloves, microcentrifuge tubes, flasks, 50 ml Falcon tubes, pipet tips (p20), tip disposal jar, paper towels, hotpads, pint-size Ziploc freezer bags, lab masking tape, Whatman 3MM paper, nitrocellulose membrane, plastic wrap, glass trays, sponges, glass pipette, glass plate (about 8 x 10), paperweight (not too heavy), hybridization bags, foil, blunt-ended forceps, biohazard bags, squeeze bottles for ddi H2O.

Reagents: Pre extracted mouse, pig, and human DNA, double deionized (ddi) H2O, restriction enzyme, restriction buffer, agarose (0.8%), 10X TAE, 20X SSC, loading dye (for agarose gels), 0.25M HCl solution, denaturation solution, neutralization solution, prehybridization solution, neutralization solution, hybridization solution, nonradioactive DNA probe (custom ordered), wash buffer, Blocking buffer, Anti-DIG-alkaline phosphate, Detection buffer, x-phosphate solution, NBT buffer, color substrate solution.

**Note**  Many of these supplies are costly, especially the larger equipment.  Austin Community College’s BioTechEd program offers an outreach program to loan supplies to high schools free of charge.  These supplies include micropipetters and electrophoresis equipment.  They might also be able to contact other labs to make other equipment available for use, as well as offer teacher support in ordering supplies.  For more information, contact Peggy Maher at 512-223-3285. 

IV. Supplementary materials, handouts: Lab protocol worksheet, images, webliographer (http://webliographer.com/humangenome)

V. Standards

Texas Essential Knowledge and Skills: 112.43.1 A and B; 112.43.2 A, B and C; 112.43.3 A

National Science Education Standards: Understandings about science and technology (content standard B),  identify questions and concepts that guide scientific investigations, design and conduct scientific investigations, use technology and math to improve investigations and communications, communicate and defend scientific argument.

 

Engagement

Student behaviors / activities

Teacher behaviors / activities

Think about the body plans of mice, pigs, and humans, and identify similarities between the three organisms. 

Listen attentively to teacher’s description of the experiment and lab journal.

 

Ask students what mice, pigs, and humans have in common and discuss their responses as a class.  The teacher will then introduce the experiment, informing the students that they will select a human gene to look for in mouse and pig DNA.

The teacher will also discuss the lab journal, stating that it should include, 1.notes over concepts and procedures, 2.a record of all protocols, also indicating which steps the student participated in, and 3.observations.  Pages should be dated and numbered.  A handout with these requirement can be given to students if desired.

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Exploration

Student behaviors / activities

Teacher behaviors / activities

 Part 1: Selecting a gene

Students will look at the chromosome map found on the webliographer page, and as a class they will choose a human genetic disease that might also be found in pigs and mice based on the similarities identified by the class.  They will find out what protein is affected, then look up the sequence of the gene that codes for that protein (see webliographer for link to GenBank `99). This will be the gene they will probe for.

 

Students will participate in a discussion to decide on a hypothesis as a class, which they will record in their lab journals.

 

At this point, the students will divide into groups at the teacher’s discretion.  Two will be assigned mouse DNA, two pig DNA, and two human DNA (control).

 

Part 2: Restriction digest

 

Students will answer any questions asked by the teacher, and they will take notes in their lab notebook.

 

Students should follow along in the lab protocol while the teacher is reviewing, recording any special notes in their lab journals.

 

Students should practice using the micropipettes with water before beginning the experiment.

 

In groups, the students should perform steps as directed by the lab protocol, and record all procedures and observations in their lab journals.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Part 3: Gel Electrophoresis

 

In groups, the students will cast an agarose gel, load DNA, and perform electrophoresis according to protocol.   The student should also answer questions asked by the teacher and record notes, procedures, and observations in their lab journals.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Part 4: Southern Blotting

 

In groups, the students will assemble a Southern blot according to protocol.  The student should also answer questions asked by the teacher and record notes, procedures, and observations in their lab journals.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Part 5:  Hybridization

 

In groups, the student will perform DNA hybridization according to the protocol.  Since the teacher will also perform some steps due to time constraints, students should review these steps along with the teacher to grasp their purpose.  During any free time, the group should meet and work on the lab report.

The student should also answer questions asked by the teacher and record notes, procedures, and observations in their lab journals.

 

Part 1: Selecting a gene

The teacher will offer advice to the class in selecting a gene to probe for.  Good criteria might include a protein with only one form, as well as a short gene sequence. 

The teacher will also order nonradioactive probes for this gene from a company specializing in this.

 

At this point the teacher will ask the class if they know what a hypothesis is, making sure they understand that it is the prediction they will be testing in their experiment.  The teacher will guide the class with questions to agree on a justified hypothesis about whether or not the mouse and pig share the human gene, and why or why not.

 

The teacher will break the class into groups and assign them the type of DNA to probe.  It is important that the students realize the human trial is the control in the experiment. 

 


 
Part 2: Restriction digest

Before beginning, the teacher will briefly review lab safety that had already been discussed at the beginning of the year, as well as the importance of careful handling of materials and accurate measurement during this experiment.  Emphasize that students should wear latex gloves at all times.

The teacher will give an introduction to restriction enzymes (by lecture and asking questions to draw upon students prior knowledge and reasoning skills) that includes the following information: 1. Restriction endonucleases are bacterial enzymes that cut DNA at certain short sequences that they recognize.  2. Why do bacteria have these enzymes? –to destroy foreign DNA from something that might harm the bacteria.

The teacher will then review protocol handed out to students, and also give a demonstration on using the micropipettor.  Ask the students to pick them up and find the two stops when pushing down the plunger: the first is the point to which you push when drawing up liquid, and the second is the point to which you push to eject liquid out of the tip.  Instruct each student to practice putting tips on the pipetter and practice measuring with water. 

During the activity, the teacher will circulate around the room, monitoring each group’s activities and offering assistance when needed.

Part 3: Gel Electrophoresis

As in the previous part of the experiment, the teacher will give an interactive introduction to gel electrophoresis.  The following information should be conveyed to the students: 1. An agarose gel is a solid form in which the agararose particles are scattered with spaces between them.  When surrounded by electrophoresis buffer (containing electrolytes), it will conduct a current through it when a power source is applies.  2.  DNA has a negative charge.  What would happen if DNA was placed in a gel and current applied?—the negative charge would be attracted to the electric current, and the DNA would move down the gel with the current.  3.  In the last lab, the DNA was cut into pieces—are all these pieces the same size?  Will the large pieces and small pieces travel at the same speed down the gel? --The larger pieces have a harder time moving through the spaces between the agarose particles, so they move more slowly.  Small pieces move fast, and the DNA will separate according to size.

Before class:  The teacher will need to prepare TAE and agarose (see protocol).  The agarose solution will need to be kept at 65˚C in a waterbath.

During first day, the teacher will review the protocol with the students, giving a demonstration of how they should set up the pouring tray.  The teacher will also warn students 1. not to burn themselves while pouring the gels, and 2. to use caution when handling the fragile gels, as they will tear easily.

During the activity, the teacher will assist students in pouring their gels, making sure each apparatus is set up correctly before pouring.  After checking for proper storage, the teacher will also transport gels to the refrigerator to be stored in for the next day.  The teacher will also assist students to prepare the DNA for loading, making sure all are wearing gloves while handling the loading dye. The teacher will warn students of its toxicity (formamide is a terratogen, which mutates the sex cells).

During the second day, the teacher will have the gel boxes assembled at each group’s station in order to save time.  The teacher will also have the waterbath ready at 95˚C so the students can denature the samples they prepared earlier, as well as have ice available for after the denaturing. 

The teacher will review the loading protocol with the students, and also emphasize caution when using the power supply.  Assist students in loading gels, instructing them to eject the pipet tip slowly and carefully so that all of the sample goes into the well.  Before turning on the power supply, check each groups wells to ensure proper loading.  Also check the running voltage and wattage to make sure it is at the correct settings.  The teacher will likely have to turn off and store the gels after class since they will have to run longer than the class period.

Part 4: Southern Blotting

As in the previous part of the experiment, the teacher will give an interactive introduction to Southern Blotting, using the graphic image as a resource.  The following information should be conveyed to the students: 1. Southern blotting transfers the DNA from the gel to a nitrocellulose membrane, where the DNA can be fixed and analyzed.  2.  The transfer happens by capillary action of the buffer moving up through the layers, carrying the DNA to the membrane.  3. How do we get the DNA to stick to the membrane?--first denature it (make it single stranded before blotting), then bake it in a vacuum oven. 

While the gels are running on the day before the previous day, the teacher will review the protocol and supplies needed for Southern blotting, giving a demonstration of what the students will do.  Warn the students to use caution when handling the gel and them membrane: they both tear easily.  Also the membrane should be handled with clean forceps, not gloves. 

On the day of the activity, the teacher will circulate throughout the room to check each group’s setup and answer any questions the students may have.

Part 5:  Hybridization

As in the previous part of the experiment, the teacher will give an interactive introduction to DNA Hybridization that should include the following information.  1.  DNA hybridization is the joining of two comlementary strands, in this case the DNA from the gel and the single stranded probe.  2.  The probe is designed so that it is complementary to a section of the gene we’re looking for.  3.  How do you see the probe?—It has receptors that bind to antibodies.  After soaking the membrane in the probe that will bind to the gene, it is soaked in the antibodies that will bind to the probe.  These antibodies can then be detected by adding a coloring agent. 

This section of the lab is extensive and will take three days to complete.  The teacher will need to complete steps that the student will not have time to do (see protocol).  The teacher will also need to prepare solutions for use by the students.   

During the lab, the teacher should circulate throughout the room to check each group’s progress and answer any questions the students may have.  Also advise the students to work on their lab reports during any lag time (while waiting for the membrane to finish soaking).


 

Explanation

Student behaviors / activities

Teacher behaviors / activities

Students will answer questions about that day’s activity, follow along with the lab protocol, and record notes and steps in the lab journal.

 Before each section of the lab exploration, the teacher will review concepts, protocols, and safety measures with the students.


 

Elaboration

Student behaviors / activities

Teacher behaviors / activities

 Each lab group will briefly share their results with the class (3-5 minutes) and state whether they are able to reject or accept their hypothesis.  They will then participate in a discussion over how this activity fits into the Human Genome Project.  They should think about the number of genes shared with organisms like the roundworm and the chimpanzee, and think about what this means in terms of evolution.

 After group presentation of results, the teacher will state that 50% of our DNA is the same as a roundworm, and 99% is the same as a chimpanzee.  To start a discussion, ask what the students think this means in terms of evolution.  How might knowing the gene sequences of other animals help the human race?  Does it change our view of the human species and its place in the world? 


 

Evaluation

Student behaviors / activities

Teacher behaviors / activities

 The students will keep a chronological lab journal, recording any notes given for the day, the steps in the experiment conducted that day, a record of what the student does, and any significant observations. 

Each group will also submit a lab report, composed of 5 sections: 1. introduction, 2. hypothesis with predictions, 3. methods, 4. results, 5. conclusion.  The first three sections will be submitted as a rough draft, and the final paper will include all five sections. 

Students will also present their findings to the class in a brief presentation near the end of the nine weeks.

 The teacher will collect lab journals once per week during the lab investigation and comment on what the student is doing correctly and what areas need improvement. 

The teacher will hold a meeting with each group to give feedback after reviewing the rough draft.  During this meeting, the results and conclusions will be discussed to make sure the students are heading in the right direction with their final papers.

The teacher will evaluate the papers based on how well they interpret their results in relation to the hypothesis, how accurately they reflect the methods, and how well they documented the results of the experiment with descriptions and pictures.

 


 
 

 

RESTRICTION DIGEST PROTOCOL

 

Restriction endonucleases (or enzymes) recognize short DNA sequences and cleave double stranded DNA at specific sites within or next to the recognition sequences.  A restriction digest has many different applications in molecular biology, but for our purposes, we must cut the whole DNA into smaller fragments so that they will migrate down an agarose gel during electrophoresis in the next section.

 

Materials needed:

DNA Sample

10X restriction enzyme buffer (should be available with enzyme)

Restriction enzyme (EcoRI is listed, but if a recognition sequence is present within the  gene you’re probing for, you’ll want to pick a different one with a different recognition sequence)

Double-deionized (ddi H2O)

Waterbath

Access to a freezer

 

Procedure:

  1. Pipette into a clean microcentrifuge tube:          
    1. X μl DNA
    2. 2 μl 10X restriction buffer
    3. 18-X μl H2O

The concentration of DNA may vary, and the volume to be added will be designated by your teacher.  The amount by weight should be about 4 μg of DNA.

 

  1. Add restriction enzyme EcoRI to the DNA mixture.  You should add 1 to 5 U per μg of DNA.  The restriction enzyme degrades at room temperature, so store on ice when in use and in the freezer when not in use!!!

 

  1. Incubate at 37˚C for 45 minutes. 

 

  1. To stop reaction, put the tube in the freezer.

 

 

GEL ELECTROPHORESIS PROTOCOL


Background


In the lab introduction, you learned that electrophoresis (a term which literally means "to carry with electricity") is a technique for separating and analyzing mixtures of charged molecules. Clearly this separation wouldn't work very well if the molecules were just sprinkled on the surface of the gel box fluid! Instead, the mixture to be separated is "loaded" into slots or "wells" of a slab of jelly-like material called agarose. Agarose is a very pure form of agar, which is actually made from a kind of seaweed.

 

To prepare or "cast" an agarose gel, agarose powder is mixed with buffer, heated, and poured into a casting or gel tray containing a comb. When the gel has cooled and solidified, the entire casting tray is lowered into the gel box and covered with buffer, which allows the electricity to flow and prevents changes in pH. The comb is removed, creating empty wells. Then, a micropipettor is used to place a small amount -- usually just a few microliters -- of the mixture to be separated into each well.

In order to track where the "invisible" DNA runs on a gel, we add two dyes to the DNA sample. One dye runs slightly faster and farther than DNA; the second dye runs slower and not as far as the DNA.

 

Materials per team

Power supply             

buffer [1X TAE]      

glass beaker, 50 ml, for agarose

gel box with gel tray

loading dye

beaker, 500 ml, for buffer

P-20 micropipette & tips

container for waste (tips)

agarose, [0.8%], melted & kept hot in a 65 degree C bath or incubator.

Waterbath

ice

 

Solution preparation (by teacher):

 

1X TAE:  This buffer usually comes in 10X concentration, so dilute 1 part buffer to 9 parts ddi H2O for use with electrophoresis.

 

Agarose:  Mix one-two batches of 0.8% agarose in 1X TAE buffer (not water!). For example, 400 mL of 0.8 % agarose would be enough for 15-20 teams, each using 20-25 mL. To make this, mix 3.2 g of agarose power in 400 mL of 1X TAE buffer.  Microwave the solution at 30 second intervals until the agarose completely dissolves when swirled.  Place the beaker in a 65˚C waterbath to keep in liquid state until students are ready to pour their gels.




Part 1: Casting a Gel

 

1. Loosen the screws at the ends of a casting tray, if necessary, to raise the "gates" at each end; then, tighten the screws (not too tight) until there is enough tension to hold the gates in place. If the gel tray does not have gates, apply tape across the ends of the tray so that it will hold liquid.  Insert a comb in the end slots of the empty tray. The teeth of the comb should not touch the bottom of the tray!   See diagram.

  1. Place the prepared casting tray on a paper towel.
  2. Obtain a beaker with 25-30 mL of liquid agarose, which has been kept at 65-70˚C in a water bath. Pour the agarose evenly into the casting tray. DO NOT POUR THE GEL IF AGAROSE IS ABOVE 70˚ C. Clean and dry the beakers immediately after pouring, making sure that none of the agarose is left.
  3. DO NOT JAR or MOVE the casting tray as the gel solidifies. This ensures a smooth, even gel. As the agarose polymerizes (about 10-15 minutes), it changes from clear to slightly opaque.
  4. Label a Ziploc baggie with your group number.  When your gel has set, carefully remove your gel from the tray and place it in the baggie with enough TAE to cover it (ask the teacher if you’re not sure).  Make sure your bag is sealed and carefully place it in the designated spot in the refrigerator.  Use extreme caution when handling your gels, as they tear easily.

Part 2: Sample preparation:  DNA samples are added to a loading buffer to keep them denatured (single stranded) while running through the gel, as well as for visualization purposes.

  1. In a clean microcentrifuge tube, add 10 µL DNA and 10 µL of loading buffer (containing dye).  USE GLOVES WHEN HANDLING LOADING BUFFER—IT CONTAINS A TOXIC CHEMICAL CALLED FORMAMIDE.
  2. Mix the DNA and buffer by pipetting up and down 10-20 times with the micropipetter. 
  3. Store this sample labeled with your group number in the refrigerator until ready to load.
  4. Before loading, denature the sample in a 95˚C waterbath for 10 minutes, then chill immediately on ice.  The quick chill prevents the DNA from re-forming double strands. (TEACHER WILL DO THIS FOR YOU)

 

Part 3: Electrophoresis

  1. Fill the electrophoresis box with about 300 mL of 1X TAE electrophoresis buffer. Orient the gel box so that the wire leads are facing you.

 

  1.  Submerge the tray onto its platform in the gel box. The comb should be located at the cathode end (black lead; (-) end). Remember DNA RUNS TO RED, so it has to start (in the well) at the black end.  The level of the buffer should be only a few mm above the surface of the gel.

 

  1. Carefully remove the comb from the gel (pull it straight out). You'll notice that this left behind six little empty "slots" or wells in the gel. To check to see if there is enough buffer, look to see that there is no "dimpling" of the buffer above the wells. Add more buffer if needed.

 

Loading the Gel

  1.  With your gel lined up in its box with the wells to your left, the contents of Tube "1" should be loaded in the well closest to you. Thus, when the gel is turned so that the wells are at the top, "1" will be in the left-hand corner.

  1. Draw 2 µL of DNA/loading dye mixture into a p20 micropipettor. (Remember: depress the plunger to the FIRST STOP before lowering the tip into the sample.)
  2. Steady the pipet over the well, using your second hand to support your pipetting hand or arm.  
  3. Lower the tip of the pipet under the surface of the buffer directly over the well -- but do not lower the tip into the well itself, or you risk puncturing the bottom of the gel.

 

  1. Gently depress the pipet plunger to slowly expel the loading dye into the well. If the tip of the micropipet is centered over the well, the dye will sink to the bottom of the well.

REMEMBER - keep the pipet plunger depressed to the SECOND STOP until the pipet tip is out of the gel box or you'll draw your sample back into the tip!



Electrophoresis of the DNA

  1. Close the top of the gel box and connect electrical leads anode to anode (red to red) and cathode to cathode (black to black). Both electrodes should be connected to one power supply channel.
  2. Set the power supply to approximately 100 V, and turn it ON. (As a check to see that electricity is flowing, look for bubbles at the wire at either end of the gel box.)
  3. Shortly after the voltage is turned on, you should see the dyes slowly moving through the gel toward the positive side of the gel box.
  4. Electrophorese until the blue band (Bromphenol blue) is almost at the end of the gel. 
  5. Turn off the power and disconnect the leads.
  6. Place your gel back in the baggie labeled with your group number and enough TAE to cover it.  Store in the refrigerator over night. 

NOTE: Due to the short class periods, your teacher will likely have to stop the power and remove the gels from the boxes for you.   Make sure your baggie is labeled and next to your gel box so that you get the correct gel back the next day.

 

 

SOUTHERN BLOTTING PROTOCOL

 

In order to probe DNA, it has to be fixed onto a membrane first (we are using a nitrocellulose membrane).  DNA cannot be probed on an agarose gel.  The southern blot takes advantage of capillary action to transfer the DNA molecules from the gel to the nitrocellulose membrane.  Remember that nitrocellulose must be handled with care because it is rather brittle.

 

 

Materials: Shaker platform, ddi H2O, denaturation solution, neutralization solution, glass baking dish, 20X SSC buffer, nitrocellulose membrane, sponges, paper towels, Whatman 3MM paper, saran wrap,  glass pipette, glass plate, paperweight, blunt-edged forceps, scissors, pencil, vacuum oven.

Procedure:

Preparing gel (done by teacher):

1.      Rinse the gel in ddi H2O and place in a clean tray containing ~ 10 gel volumes worth of 0.25M HCl.  Shake slowly on a platform shaker for 10 minutes at room temperature.

2.      2.  Add ~ 10 gel volumes worth of denaturation solution and shake for 30 minutes at room temperature.

3.      Pour off the denaturation solution and rinse the gel with distilled water.  Add ~ 10 gel volumes worth of neutralization solution and shake for 30 minutes. 

The HCl changes the chemistry of the DNA, allowing the DNA strands to be denatured by the denaturation solution (they “unzip” down the middle and become single-stranded).  Only single stranded nucleic acids bind to the membrane used in the Southern blot. In addition, the DNA must be single stranded to be able to base-pair (hybridize) with the probe. The neutralization solution brings the pH to <9 so that the DNA will bind to the membrane used in the next part of the experiment.

 Southern blot:

  1. Place an oblong sponge, slightly larger than the gel, in a glass or plastic dish.  Fill the dish with enough 20X SSC buffer to leave the soaked sponge about half-submerged in buffer.
  2. Cut three pieces of Whatman 3MM paper to the same size as the sponge.  Place these on the sponge and wet them with 20X SSC buffer.
  3. Place the gel on the filter paper and squeeze out air bubbles by gently rolling a glass pipette over the surface.
  4. Cut four strips of plastic wrap and place over the edges of the gel to frame it.  This is to prevent the buffer from “short-circuiting”—i.e. so that it flows through rather than around the gel.
  5. Cut a piece of nitrocellulose membrane just large enough to cover the exposed surface of the gel.  Avoid handling the membrane even with gloved hands—use clean blunt-ended forceps instead.
  6. Pour distilled water into a separate glass dish to about 0.5 cm deep.  Submerge the nitrocellulose membrane, then place it in another dish filled with 20X SSC for 10 minutes.
  7. Place the wetted membrane on the surface of the gel.  Try to avoid getting air bubbles under the membrane; remove any that appear by carefully rolling a glass pipette gently over the surface.  It is important to lay the membrane down precisely the first time, because DNA starts moving across the membrane immediately.
  8. Puddle the surface of the membrane with 20X SSC.
  9. Cut five sheets of Whatman 3MM paper to the same size as the membrane and place these on top of the membrane.
  10. Cut paper towels to the same size as the membrane and stack these on top of the Whatman 3MM papers to a height of about 4 cm.
  11. Lay a glass plate on top of the structure and place a weight on top to hold everything in place. 
  12. Leave overnight.  The liquid will soak from the tray to the dry paper towels on top, going through the gel and transporting the DNA to the membrane.
  13. The next day, carefully remove the paper towels and filter papers to recover the membrane.  Mark in pencil the position of the wells on the membrane, and ensure that the up-down and back-front orientations are recognizable.  Do not use ink, as it may wash off.
  14. Rinse the membrane in 5X SSC, then place it on a sheet of Whatman 3MM paper.  This rinse removes agarose fragments and excess salt that may cause problems during hybridization.
  15. To immobilize the DNA on the membrane, place the rinsed membrane on a piece of Whatman 3MM paper and bake in a vacuum oven at 120˚C for 15 to 30 minutes.  The oven must be a vacuum oven, or the nitrocellulose membrane will spontaneously combust.

HYBRIDIZATION PROTOCOL

In the last section, you performed a Southern blot to transfer the DNA from the agarose gel to a nitrocellulose membrane for probe analysis.  This membrane will be pretreated with a prehybridization solution to make the DNA receptive to the probe.  The probe is then added (in a hybridization solution) to the membrane and incubated in the hybridization oven.  This oven regulates the temperature as well as shakes the membrane to ensure thorough coverage by the solution.  If the gene we’re looking for is present on the membrane, the labeled DNA probe (which is complementary to a region of the gene), will bind to the gene.  The membrane will then be washed in an antibody solution that binds to the probe.  This antibody can be detected with an agent that will change color where the probe is locate.

Day 1:

  1. Place the dried membrane in a hybridization bag containing 20 ml prehybridization solution per 100 cm2 of membrane.  Seal the membrane, and prehybridize at 50˚ to 60˚C for 2 hours in hybridization oven (temperature may vary depending on probe used). 
  2. Denature the probe for 10 minutes at 95˚C, then chill on ice.  (teacher does this)
  3. Dilute the probe in hybridization solution (concentration will depend on the probe). (teacher does this )
  4. Discard the prehybridization solution from the bag and add the hybridization solution (at least 20 mL per 100 cm2 of membrane).  (teacher does this)
  5. Hybridize overnight at 50˚ to 60˚C in the hybridization oven (temperature may vary depending on probe used). (teacher does this )
  6. When hybridization is done, save the leftover solution (containing unused probe) and freeze at -20˚C.  It will last for one year. (teacher does this)

Day 2:

  1. Wash the membrane twice, 5 minutes per wash, in 2X wash solution at room temperature. 
  2. Wash the membrane twice, 15 minutes per wash, in 0.5X wash solution at room temberature.  These four washes remove unbound probe from the membrane that will cause background when detecting the probe that has bound to the DNA.
  3. Store the membrane overnight in washing buffer at 4˚C.

Probe detection:

  1. In a clean dish, agitate the membrane gently in Blocking solution for 30-60 minutes.  (TEACHER DOES THIS)
  2. During the end of this time, prepare the antibody probe detector solution.  Dilute the Anti-DIG-alkaline phosphate (antibody) 1:5,000 in Blocking buffer for a working concentration of 150 mU/ml.  Mix gently by inversion.  For example, add 6μl antibody to 30ml of Blocking solution.  This is usable for 12 hours at 4˚C. (TEACHER DOES THIS)
  3. Pour off the Blocking solution and incubate for 30 minutes at room temperature, making sure the solution is shaking. (TEACHER DOES THIS)
  4. Discard the Blocking solution with antibody.  Wash the membrane twice, 15 minutes per wash, in 15 mL Washing buffer.  These washes remove unbound antibody. (TEACHER DOES THIS)
  5. Mix 45μl NBT solution and 35μl X-phosphate solution (antibody detectors) in 10μl of Detection buffer.  Wrap the tube in foil, as the solution is light sensitive.  (TEACHER DOES THIS)

 

Day 3:

  1. Equilibrate the membrane in 20mL Detection buffer for 2 minutes.
  2. Pour off Detection buffer, and add 5 ml color Substrate solution to the membrane.  Incubate the membrane in a sealed plastic bag or box in the dark.  Do not shake the container while the color is developing.  Make sure the membrane is oriented so that the DNA is facing up.  The color starts to appear within a few minutes, and the reaction will be complete in about 12 hours.
  3. At about five minutes till the bell, take a picture for your records while the gel is still submerged.
  4. Wash the membrane with H2O to stop the development. 
  5. Take another picture for your records.
  6. Dry the membrane at room temperature.  Make sure you mark the tray with your group number!  After the membrane is dry, take yet another picture.