1. Carry out PCR for 20, 25 and 30 cycles. Analyse the PCR fragments by agarose gel electrophoresis. Find out how the number of cycles affects the amount of DNA generated. Observe that the DNA fragments generated from each plant species are all the same size. Aims of this case study

2. The primers have been designed to match non-coding regions of DNA between genes. These regions have a higher frequency of mutation. Plant species that are related will tend to have fewer mutations. Species that are evolutionary distant are likely to have more mutations. These differences will show up in the size of fragments obtained through PCR. Background information regarding the primers Variable non-coding region Highly conserved region of DNA Primer 5 3 5 3

3. Purification of the Extracted DNA Try to choose areas that have the same degree of staining. This will remove inhibitors of PCR.

4. 1. Place a clean cutting/backing board behind the absorbent layer. Place the tip of the punch over the area to be sampled, press firmly and rotate to remove a paper disc. Choose an area where the extract has soaked through to the back. Clean the punch between samples by removing a disc of paper from an extract-free area. Purification of the Extracted DNA

5. 2. Use a cocktail stick to transfer the disc from the punch into a labelled, clear 1.5-ml micro centrifuge tube. Use a different cocktail stick for each sample. Purification of the Extracted DNA

6. The next two slides are about micropipettes 1-ml syringes or narrower micro syringes can be fitted with a disposable graduated tip. White tips are marked 2 and 10 μl. Yellow tips are between 10 and 100 μl. A short length of silicone rubber tubing can be used to fix the tip to the syringe to give an effective seal.

7. You may be lucky enough to have adjustable pipettes. Twist the dial to the desired volume. Pick up the pipette tip. Press the plunger to the first, soft, stop. Insert the pipette tip into the solution to be transferred. Slowly release the plunger to retrieve the liquid. Move the pipette tip to above the desired well. Press the plunger past the first stop to the second, hard, stop to transfer the liquid.

8. 3. Add 150 l of purification reagent to the disc. Use different tips for different discs. 4. Close the tube and flick it to wash the disc. Ensure the disc remains in the liquid. 5. Remove and discard the purification reagent. 6. Repeat steps 3, 4 and 5. Purification of the Extracted DNA

9. 7. Use a fresh tip to add 150 l of TE-1 buffer to the disc. Use different tips for different discs. 8. Close the tube and flick it to wash the disc. Ensure the disc remains in the liquid. 9. Remove and discard the buffer. 10. Repeat steps 7, 8 and 9. Purification of the Extracted DNA

10. Amplification of Chloroplast DNA (cpDNA) 1. Label the PCR tube. 2.– 4. Add reagents: 4 l sterile deionised water 10 l primer 1 (CHc) (yellow) 10 l primer 2 (CHd) (blue) PCR beads contain Taq poymerase, dNTPs, buffers and co-factors.

11. 5. and 6. Flick the bottom of the PCR tube – centrifuge if necessary. 7. Use a clean cocktail stick to transfer the disc from the micro tube to the PCR tube. Ensure the disc is submerged in the PCR reagents. Amplification of Chloroplast DNA (cpDNA)

12. 8. Place the PCR tube in the thermal cycler. Amplification of Chloroplast DNA (cpDNA) Alternatively use three water baths set to the correct temperatures.

13. 9. Samples may be frozen ready for gel electrophoresis. Amplification of Chloroplast DNA (cpDNA) The PCR programme 1.94°C for 2 minutes to ensure maximum separation of the strands. 2.30 cycles of the following steps: 94°C for 30 seconds 55°C for 30 seconds 72°C for 45 seconds 3.(72°C at final stage for 2 minutes)

14. Gel Electrophoresis of PCR Products If a DNA sizing ladder is available add 2 l of loading dye to 8 l of DNA ladder (lilac micro tube). Mix and load all 10 l into well 1 in a 1.5% agarose gel. This is not on your work card so dont worry. Diagram: Dean Madden NCBE

15. 3. Repeat step 3 for other PCR samples. 2. Load 10 l of the sample into a different well in the gel. Note sample number and well. 1. Using a fresh tip, add 2 l of loading dye to your amplified sample and mix. Gel Electrophoresis of PCR Products

16. Diagram: Dean Madden NCBE Gel Electrophoresis of PCR Products Fit the carbon-fibre electrodes as shown.

17. Gel Electrophoresis of PCR Products Attach the power leads. The samples run from black to red.

18. Gel Electrophoresis of PCR Products You will need stain, either Azure A or Fast Blast. You will also need a suitable tray, such as a shallow ice-cream tub, to hold the gel and a piece of plastic to help move the gel.

19. Gel Electrophoresis of PCR Products Using the plastic card, gently scoop the gel from the tank into the tray.

20. Gel Electrophoresis of PCR Products 4. and 5. Pour Azure A or Fast Blast stain over the gel and leave for 4 minutes before de-staining. Remember not to pour the stain down the sink!

21. Your gel may look something like this........... Gel Electrophoresis of PCR Products The gel was stained with Azure A. Lanes 1–3 spinach 1–20 PCR cycles 2–25 PCR cycles 3–30 PCR cycles Lanes 4–6 chard 1–20 PCR cycles 2–25 PCR cycles 3–30 PCR cycles