• provide evidence for assessment of Outcome 3
  (For advice on marking Outcome 3 report, please contact the SAPS Scotland office.)
  
• develop knowledge and understanding of cutting DNA with restriction enzymes
  
• develop problem solving skills and in particular Outcome 2 PCs:
  (c) conclusions drawn are valid and explanations given are supported by evidence
  (d) experimental procedures are planned, designed and evaluated appropriately

This experiment is done with the help of the Plant DNA Investigation kit obtained from NCBE, Science and Technology Centre, Earley Gate, University of Reading, Whiteknights, READING, RG6 6BZ. Tel: 0118 987 3743 Fax: 0118 975 0140. Cost £130.00 (2005 prices). SAPS offers sponsorship towards the initial cost of a kit providing that a teacher from the school has attended a SAPS DNA workshop. Contact SAPS at Edinburgh (Tel: 0131 558 8212) or at Cambridge (Tel: 01223 507168) to obtain the appropriate form. Refills and individual items can also be obtained from NCBE. Student Guides and a Technical Guide are supplied with the kit and these supply a great deal of relevant background information.

In this experiment it is assumed that a 4-tooth gel comb is used to provide 4 wells in each gel. If using a 6-tooth gel comb the wells hold less DNA.

In the experiment, a batch of DNA is digested by two different restriction enzymes. Due to inappropriate buffer concentrations, the activity of the second enzyme will be reduced. However, evidence of its activity should still be apparent.

Following these instructions, this experiment requires three separate days to be completed.

Day 1 - Practising with the microsyringe and digesting the DNA requires 30-40 minutes followed by a 40 minute incubation at 37°C. After the incubation the small tubes should be stored in the freezer until the next day.

Day 2 - Separating the DNA fragments requires about 30 minutes to set up. Ensure the gels are loaded close to the electricity supply so they do not have to be moved once loaded. As long as the electric current has been applied long enough for the DNA to have moved out of the wells (40-50 minutes at the lowest voltage) the electricity can be switched off and on as required (however, when switched off, loading dye will diffuse out of the gel making it difficult to see how far the DNA fragments have travelled).

Day 3 - Staining the gels requires only 5-10 minutes but it can take another 15-20 minutes to identify any visible bands in the gel and measure the distance each band has travelled.

The following table is a guide to suitable power supplies, voltages and total lengths of time to apply voltage to obtain good separation of DNA fragments.

CARE! Students hands should be DRY when carrying out the electophoresis.

In order to satisfy the core skill in problem solving, students will be required to identify and obtain resources required for themselves. Further advice on supply of material is given in the Technical Guide.

Investigating Plant DNA - Student Guide and Technical Guide. These booklets accompany the DNA kit available from NCBE.

Micklos, D. and Freyer, G. (2003) DNA Science. A first course. Cold Spring Harbour Laboratory Press 2nd Ed ISBN: 0879696362

Miller, M.B. (1993) DNA technology in schools: a straightforward approach, Biotechnology Education, 4(1), 15-21.

Miller, M.B. (1994) Practical DNA technology in school, Journal of Biological Education, 28(3) 203-211.

Miller, M.B. and Russell, G.A. (1996) Practical DNA technology in school - 2: Computer analysis of bacteriophage lambda base sequence, Journal of Biological Education, 30(3) 176-183.

www.ncbe.reading.ac.uk

This experiment was produced by the SAPS Biotechnology Scotland Project. Funding for the project was provided by SAPS, Unilever and The Scottish Office. Support was also provided by Edinburgh University, Quest International, the Scottish CCC, the Higher Still Development Unit and SSERC.

2 pink tubes containing the restriction enzyme EcoR1

2 green tubes containing the restriction enzyme Hind111
1 yellow tube (empty)
1 white tube of DNA suspension
1 microsyringe and 6 tips
1 float
1 vial loading dye
1 piece of parafilm
1 marker pen

electrical supply (see Teacher/Lecturer Guide)
2 electric wires with crocodile clips
enzyme tubes in the float from the previous lesson
vial of loading dye
gel in a plastic tank with comb, covered in buffer solution
microsyringe and 4 tips
piece of black card
2 pieces of carbon fibre tissue

tank containing your gel from previous lesson
stain (10 cm³)
gloves
eye protection

Day 1 - waterbath at 37°C
Day 2 - bottle of TBE buffer

Rehydrating the DNA - The l (lambda) DNA in the narrow white tubes provided in the Plant DNA kit must be rehydrated with distilled water shortly before the experiment is carried out. Follow the instructions on page 10 of the Student Guide provided with the kit. One tube of DNA is required per group of students.

Preparing the agarose gel - If necessary, this can be done a few days before the experiment is carried out. Follow the instructions on page 12 of the Student Guide. One gel is required per group of students.

Two pieces of carbon fibre electrode tissue (approximately 42 mm x 22 mm) are required per group. Wear gloves when handling the carbon fibre tissue.

Dilute 1 volume of the TBE buffer concentrate with 9 volumes of distilled water. About 35 cm³ will be required per group (11-12 cm³ to dissolve the agarose and form the gel and the rest to cover the gel once it is set). The liquid can be reused for 3-4 ‘runs’ after which it should be discarded.

Dilute the concentrated stain for DNA with an equal volume of distilled water. About 10 cm³ of stain is required per group. This diluted stain can also be reused several times.
Wear gloves and eye protection when handling the stain.

Recipes for the various buffers and dyes used in the experiment are given in the Technical Guide supplied with the kit.

Making a float - Make 4-5 holes in a plastic petri dish lid or base using a small hot rod. The holes should be about 8 mm in diameter. This will allow the pointed end of the microtubes through but will hold their top end. Alternatively, the holes can be made in a thin piece of foam e.g. camping mat.

Pieces of parafilm (about 5 cm x 5 cm) are required for the microsyringe exercise. However, any non-absorbent paper e.g. benchcoat will be suitable.

9 volt PP3 batteries can be obtained very reasonably (£1.34 each - 2005 prices) from Technology Supplies Ltd., Phoenix House, Tern Hill, Market Drayton, Shropshire TF9 3PX Tel: 01630 637300 Fax: 01630 637302

It is not appropriate to provide all equipment and materials in, for example, a tray system for each student/group. Equipment and materials should be supplied in a way that students have to identify and obtain resources. Normal laboratory apparatus should not be made available in kits but should generally be available in the laboratory. Trays could be provided containing one type of specialist equipment or materials.

All microtubes and gels can be safely disposed of in the bin. Buffer, loading dye and stain can be diluted and washed down the drain. A fuller account of safety is covered in the Technical Guide accompanying the kit.

Read through the Student Activity Guide and consider the following questions.

1. What is the aim of the activity?

2. What measurements are you going to make?

3. Are you familiar with how the restriction enzymes act on DNA?

4. Are you aware of what is happening during electrophoresis?

•  What safety measures are you required to take?

•  Are you familiar with the microsyringe and how to deliver a set volume using it?

Prepare a table with suitable headings and units to record the number of base pairs in each identified DNA fragment and the distance it has travelled through the gel.

1. Why are some DNA fragments not visible?

2. Why have some DNA fragments not separated sufficiently to be seen as separate bands?

3. Is there evidence that the DNA was not evenly distributed in its original tube? What can be done to prevent this?

4. How long should the electric current be passed through the gel so that DNA bands will be separated as much as possible?

5. Can you account for some lanes of the gel being blank?

This experiment uses most of the basic techniques involved in genetic fingerprinting. The DNA is digested or ‘cut up’ using restriction enzymes. The resulting fragments of DNA are then separated into bands using an electric current and made visible by staining.

If the order of bases in the DNA used is different each time then the number and sizes of the DNA fragments produced each time after digestion will also be different. Thus, DNA from different organisms (except clones) will give a unique result in this experiment - hence the term genetic fingerprinting.

DNA from a certain bacteriophage will be used in this experiment as only one, short chromosome is present in the phage. This will result in only a few different fragments being formed, thus making their separation into distinct bands more likely.

Nuclear DNA from animals or plants consists of many large chromosomes. After digestion, a very large number of fragments is formed. If all these fragments were stained, a smear would result. To obtain distinct bands (a fingerprint) with this complex DNA, only certain fragments are selected using probes.

The simple, bacteriophage DNA is going to be digested in 3 different ways:
- by mixing one sample of DNA with a restriction enzyme called EcoRI
- by mixing another sample of DNA with a different restriction enzyme called HindIII
- by mixing a third sample of DNA with both of these enzymes

Each restriction enzyme will cut the DNA only when a certain sequence of bases occurs e.g. the enzyme EcoR1 cuts the DNA between bases G and A only when the sequence GAATTC is present in the DNA. The other restriction enzyme used cuts the DNA at a different sequence of bases. Thus, each restriction enzyme is specific.

The number of DNA fragments formed after digestion by an enzyme will depend on the number of times the particular sequence of bases which the enzyme acts on is present e.g. the sequence GAATTC occurs 5 times in the bacteriophage DNA used in this experiment. The DNA will therefore be cut into six fragments when digested by the enzyme EcoR1.

2 pink tubes containing the restriction enzyme EcoR1
2 green tubes containing the restriction enzyme Hind111
1 yellow tube (empty)
1 white tube of DNA suspension
1 microsyringe and 6 tips
1 float
1 vial loading dye
1 piece of parafilm
1 marker pen

electrical supply
2 electric wires with crocodile clips
enzyme tubes in the float from the previous lesson
vial of loading dye
gel in a plastic tank with comb, covered in buffer solution
microsyringe and 4 tips
piece of black card
2 pieces of carbon fibre tissue

tank containing your gel from previous lesson
stain (10 cm³)
gloves
eye protection

Materials to be shared:

Day 1 - waterbath at 37°C
Day 2 - bottle of TBE buffer

Instructions

Preliminary exercise

This experiment requires you to transfer very small volumes of liquids. A microsyringe is provided for you to do this. The tips that fit on the end of the microsyringe have small ‘ridges’ on them. When the tip is filled to the upper ridge 10 µl will be delivered. The lower ridge is for delivering volumes of 2 µl. Follow the hints below when using a microsyringe.

• Before loading the microsyringe, pull the plunger out a little. This gives some extra air with which to expel the last drop of liquid.
  
• When emptying the microsyringe tip, hold it vertically and at eye level.
  
• To remove the last droplet from the tip, touch it against the inner wall of the container.
  
• Do NOT touch the point of the microsyringe tip with your fingers. There are enzymes in sweat which may contaminate and result in unwanted digestion of DNA samples.

i) Draw in 2 µl of dye and deposit as drop 1 on the Parafilm.

ii) Repeat Step 1 until you have 5 separate drops of dye.

iii) Draw in 10 µl of dye and deposit it alongside the smaller drops.

iv) Now draw all five 2 µl drops into the micropipetter tip and deposit them alongside the 10 µl drop.

v) Are the two drops the same size?

1. Sit the 4 tubes containing restriction enzymes in the float on the bench.

2. With a new microsyringe tip draw the DNA suspension into and out of the microsyringe tip several times. This results in the DNA being evenly distributed. Now transfer 20 µl of DNA to each of the TWO pink tubes containing a restriction enzyme.

3. Again with a new tip, transfer 20 µl of DNA to ONE green tube containing a different restriction enzyme. Remember to mix the DNA thoroughly before transferring it.

4. Again with a new tip, transfer 20 µl of DNA to an empty yellow tube. This tube will act as a control as here the DNA will be undigested.

5. Cap the tubes and flick the sides of the tubes with one finger until the blue colour is evenly spread throughout the liquid.

6. Place the float with the 4 tubes in a waterbath at 37°C (leaving the one remaining green tube on your bench). 7. After 10 minutes the restriction enzymes will be in solution. This will allow you to transfer the entire contents of one of the pink tubes to the remaining green tube again using a NEW tip on the microsyringe. The DNA in this green tube will now be digested by both restriction enzymes. Mark the tube with a D - for double digest.

8. Flick each tube several times to mix the contents. Put the four tubes (one pink, one unmarked green, one green marked D and one yellow) in the float back into the waterbath to incubate at 37°C for at least another 30-40 minutes.

N.B. The tubes can be left until next lesson as the restriction enzymes will become denatured after a few hours. To prevent further DNA breakdown, the tubes should be stored in a freezer overnight.

Day 2 - Separating the DNA fragments

1. If not already done, cover the gel with about 20 cm³ of buffer solution (to a depth similar to that shown in the diagram below).
Buffer solutions keep the pH stable and thus prevent unwanted breakdown of unstable molecules such as DNA.

2. Remove the comb gently from the gel to expose the wells.

3. Ensure your tank is close to your electricity supply and place a piece of black card under it to make the wells more visible.

*4. Using a new tip, draw in 2 µl of loading dye and mix this thoroughly with the undigested DNA in the yellow tube by drawing the mixture up and down in the tip several times.

*5. Draw up all the contents of the tube into the microsyringe tip and load well 1 by emptying the syringe slowly when the end of the tip is in the buffer solution and directly above the well.
N.B. The tip does not actually need to be in the well as the dense dye will make the DNA solution sink.

6. Repeat the last TWO steps marked * and load each well as follows:
N.B. use a NEW microsyringe tip each time.

Well 2 - DNA digested by restriction enzyme EcoR1 (pink tube)
Well 3 - DNA digested by restriction enzyme Hind111 (green tube)
Well 4 - DNA digested by both restriction enzymes (green tube D)

7. Put a piece of carbon fibre tissue at either end of the tank.

8. Connect the carbon tissue to the electricity supply using wires and crocodile clips. Once the electricity is switched on the negatively charged phosphates in the DNA are attracted to the positive electrode. So, make sure the positive electrode is furthest AWAY form the DNA in the wells.

9. Check with your teacher what voltage you will be using and set up the electricity supply accordingly. Switch on the electricity. The TBE buffer can evaporate during electrophoresis, periodically check the depth of the buffer and top up as required (to a depth similar to that shown in the diagram in Step 5).

The loading dye is dense and helps the DNA sink into the wells. It also allows us to track the progress of the electrophoresis. It travels through the gel ahead of all but the smallest DNA fragments and so allows us to judge when to switch off the electricity.

10. After an appropriate time (e.g. 12 hours at 9 volts; 6 hours at 18 volts) switch off the electricity, disconnect the crocodile clips and remove the pieces of carbon fibre.

Day 3 - Staining the DNA

1. Return the buffer solution covering the gel to its original container.

2. Pour about 10 cm³ of staining solution (Azure A) onto the surface of the gel and leave it for at least 4 minutes.

3. Pour off the stain into a bottle labelled ‘reused stain’.

4. Wash excess stain from the surface of the gel with tap water.

5. Do NOT leave any water on the gel after rinsing. If you do the stain will move out of the gel into the water.

If the staining solution has been used on a previous occasion you may need to repeat the above procedure. If this is necessary allow at least 10 minutes for instruction 2.

Purple bands of stained DNA will appear shortly. The smaller the fragments of DNA, the further it will have travelled through the gel. However, the smallest fragments will also take up less stain and may therefore be difficult to see. Also, fragments of similar size will move similar distances in the gel, resulting in little separation between them.

Below is a table showing the number and size of DNA fragments formed during the experiment. This is possible as the entire base sequence of the DNA in the bacteriophage used has been worked out.

7. Examine your gel and try to relate the DNA fragments listed above with the bands that have appeared in each lane. For each identifiable band measure the distance it has travelled.
Measure from the front of each well to the front end of each band.

8. Make a table with appropriate headings and units showing the number of base pairs, the log of the fragment size and the distance travelled for each band.

9. Present your results as a graph with suitable scales and axes labelled with quantities and units (put fragment size OR log of fragment size on the x-axis and distance travelled on the y-axis).