1. PCR workshop (Suitable for Edvotek kits 330, 371, 372)
Edvotek Europe

2. What are we doing today? Introduction Set up PCR reaction Load gels
Electrophoresis
Analyse results

3. Health & safety No toxic chemicals
Safe solutions and reagents used in place of research materials

4. The Polymerase Chain Reaction

5. What does PCR do? PCR makes millions of copies of DNA Uses PCR machine
A DNA photocopier!

6. Cell division
DNA polymerase duplicates DNA during cell division

7. DNA polymerase in action!
Stars show DNA polymerase bound to DNA

8. Who invented PCR?
“EUREKA!!! I stopped the car. Somehow, I thought, it had to be an illusion.
Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and surely I would have heard of it.
We would be doing it all the time.”
Kary Mullis - inventor of PCR, Nobel Prize 1993

9. Mullis’s Nobel prize speech is well worth reading

10. What is in a PCR reaction
Use 5μl from tube labelled “Template DNA”
Use 20μl from tube labelled “primers”
The PCR bead
Template DNA
The starting material in a PCR reaction.
Primers are two short pieces of DNA (0-15 bases long) that determine the region of DNA to be copied.
Nucleotides
A’s, T’s, G’s and C’s to make up the new DNA strands
Taq DNA polymerase
The enzyme that makes new DNA strands
MgCl2
Required for Taq DNA polymerase to function

11. How does PCR work? Mix the following: Template DNA Nucleotides Primers
Taq DNA polymerase
MgCl2

12. Cycle through 3 temperatures
Denature: unzips DNA
Anneal: primers bind to complementary areas of target DNA
Extend: Taq DNA polymerase fills in the blanks!

13. Lots of DNA produced
Successive cycles double amount of DNA

14. Taq DNA polymerase
Thermus aquaticus bacteria that lives at high temperature
DNA polymerase crucial to automate PCR

15. Uses of PCR

16. Forensics!
... Like TV shows...

17. Paternity testing

18. Genetic testing

19. Types of PCR kit DNA template provided (intro level)
Cat no 371 DNA fingerprinting
Cat no 372 Quick PCR
DNA template must be extracted (more advanced)
Cat no 333 or 334 chromosome kit

20. The experiment

21. Quick PCR set up Carefully transfer PCR bead to 0.2ml tube & label
Add 5ul template DNA
Add 20ul primer mix
Place in PCR machine

23. Quick PCR cycles Initial denaturation 94°C for 180 seconds
Then 20 cycles of:
94°C for 30 seconds
71°C for 15 seconds (annealing)
71°C for 15 seconds (extension)
Annealing & extension are same temperature

24. EdvoCycler
PCR machine
Easy to use
Select cat no
Programmable

25. Choose programme

26. Press to select

27. Lid will heat up

28. Then cycles start

29. Then cycles start
Programme selected
= Cat no of kit

30. Then cycles start
Number of cycles
to go

31. Then cycles start
Number of cycles
completed

32. Then cycles start
Temperature for each step

33. Then cycles start
Time in seconds for each step

34. DNA electrophoresis

35. Gel casting

36. Running buffer TAE buffer 20ml 50x buffer to 1 litre with water
Distilled water ideal but tap ok
Can be reused a few times
Store unused for future use ok

37. Agarose 0.8% Agarose 3g in 375 ml dilute buffer
Melt in microwave/autoclave
Pour when hand hot

38. Agarose
Store gels for 1-2 weeks in fridge wrapped in cling film or plastic bag
Keep any left over gels and remelt next time

39. Remove comb & ends

41. Fixed volume minipipette

42. Adjustable micropipette

43. Dry loading You can load gel dry instead of through buffer!
Otherwise load “wet” through buffer
Either way, remember
Do not puncture bottom of well
Change tips between each sample

45. HexaGel and EVT 300 power supply

46. Quick PCR kit gel Loading
After PCR reaction add 5l loading dye
Load 40 l of each sample into the wells A M B
LG1 C
LG2 D E F

47. Run gels Put on lid and attach to power supply
Run for 30 minutes at 150 volts
Or 75 volts for minutes
Check for bubbles at electrode
Run until tracking dye halfway across gel

48. After gel run stain the gel

49. Stain PCR gel MetBlue card blue side down 5 mins Weigh with gel tray
Destain in warm water mins
Leave overnight in fridge for best result
Keep long term in bag in fridge

50. Stain Gel

52. Gel storage
Can store gels in fridge before staining over weeknight or weekend
Cannot store dye kits overnight before viewing as diffuse!

53. Quick PCR result

54. Thank you PCR experiments can be carried out easily
Fun and relevant to wider world
Promote understanding