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

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

• Edvotek Europe

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What are we doing today?

• Introduction
• Set up PCR reaction
• Load gels
• Electrophoresis
• Analyse results

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Health safety

• No toxic chemicals
• Safe solutions and reagents used in place of
  research materials

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The Polymerase Chain Reaction
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What does PCR do?

• PCR makes millions of copies of DNA
• Uses PCR machine
• A DNA photocopier!

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Cell division
DNA polymerase duplicates DNA during cell division
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DNA polymerase in action!
Stars show DNA polymerase bound to DNA
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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

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Mulliss Nobel prize speech is well worth reading

• http//www.nobelprize.org/nobel_prizes/chemistry/l
  aureates/1993/mullis-lecture.html

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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 As, Ts, Gs and Cs 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
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How does PCR work?

• Mix the following
• Template DNA
• Nucleotides
• Primers
• Taq DNA polymerase
• MgCl2

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Cycle through 3 temperatures

• Denature unzips DNA
• Anneal primers bind to complementary areas of
  target DNA
• Extend Taq DNA polymerase fills in the blanks!

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Lots of DNA produced

• Successive cycles double amount of DNA

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Taq DNA polymerase

• Thermus aquaticus bacteria that lives at high
  temperature
• DNA polymerase crucial to automate PCR

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Uses of PCR
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Forensics!
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Paternity testing
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Genetic testing
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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

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The experiment
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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

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Quick PCR cycles

• Initial denaturation
• 94C for 180 seconds
• Then 20 cycles of
• 94C for 30 seconds
• 71C for 15 seconds (annealing)
• 71C for 15 seconds (extension)
• Annealing extension are same temperature

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EdvoCycler

• PCR machine
• Easy to use
• Select cat no
• Programmable

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Choose programme
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Press to select
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Lid will heat up
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Then cycles start
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Then cycles start
Programme selected Cat no of kit
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Then cycles start
Number of cycles to go
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Then cycles start
Number of cycles completed
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Then cycles start
Temperature for each step
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Then cycles start
Time in seconds for each step
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DNA electrophoresis
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Gel casting
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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

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Agarose

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

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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

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Remove comb ends
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Fixed volume minipipette
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Adjustable micropipette
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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

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HexaGel and EVT 300 power supply
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Quick PCR kit gel Loading
After PCR reaction add 5?l loading dye Load 40 ?l
of each sample into the wells
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Run gels

• Put on lid and attach to power supply
• Run for 30 minutes at 150 volts
• Or 75 volts for 40-50 minutes
• Check for bubbles at electrode
• Run until tracking dye halfway across gel

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After gel run stain the gel
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Stain PCR gel

• MetBlue card blue side down 5 mins
• Weigh with gel tray
• Destain in warm water 10-15 mins
• Leave overnight in fridge for best result
• Keep long term in bag in fridge

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Stain Gel
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Gel storage

• Can store gels in fridge before staining over
  weeknight or weekend
• Cannot store dye kits overnight before viewing as
  diffuse!

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Quick PCR result
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Thank you

• PCR experiments can be carried out easily
• Fun and relevant to wider world
• Promote understanding