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

Edvotek Europe

What are we doing today?

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

Health & safety

• No toxic chemicals• Safe solutions and reagents used

in place of research materials

The Polymerase Chain Reaction

What does PCR do?

• PCR makes millions of copies of DNA

• Uses PCR machine• A DNA photocopier!

Cell division

DNA polymerase duplicates DNA during cell division

DNA polymerase in action!

Stars show DNA polymerase bound to DNA

Who invented PCR?

Kary Mullis - inventor of PCR, Nobel Prize 1993

“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.”

Mullis’s Nobel prize speech is well worth

readinghttp://www.nobelprize.org/

nobel_prizes/chemistry/laureates/1993/mullis-lecture.html

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.

NucleotidesA’s, T’s, G’s and C’s to make up the new DNA strands

Taq DNA polymeraseThe enzyme that makes new DNA strands

MgCl2Required for Taq DNA polymerase to function

Mix the following:

•Template DNA•Nucleotides•Primers•Taq DNA polymerase

•MgCl2

How does PCR work?

++ ++ ++

Cycle through 3 temperatures

• Denature: unzips DNA

• Anneal: primers bind to complementary areas of target DNA

• Extend: Taq DNA polymerase fills in the blanks!

Lots of DNA produced

• Successive cycles double amount of DNA

Taq DNA polymerase

• Thermus aquaticus bacteria that lives at high temperature

• DNA polymerase crucial to automate PCR

Uses of PCR

Forensics!

Paternity testing

Genetic testing

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

The experiment

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

Quick PCR cycles

• Initial denaturation 94°C for 180 seconds

• Then 20 cycles of:94°C for 30 seconds71°C for 15 seconds (annealing)71°C for 15 seconds (extension)

• Annealing & extension are same temperature

EdvoCycler

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

Choose programme

Press to select

Lid will heat up

Then cycles start

Then cycles startProgramme selected = Cat no of kit

Then cycles start

Number of cycles to go

Then cycles start

Number of cycles completed

Then cycles start

Temperature for each step

Then cycles start

Time in seconds for each step

DNA electrophoresis

Gel casting

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

Agarose

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

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

Remove comb & ends

Fixed volume minipipette

Adjustable micropipette

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

HexaGel and EVT 300 power supply

Quick PCR kit gel LoadingAfter PCR reaction add 5l loading dye

Load 40 l of each sample into the wells

A

M

B

LG1

C

LG2

D E F

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

After gel run stain the gel

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

Stain Gel

Gel storage

• Can store gels in fridge before staining over weeknight or weekend

• Cannot store dye kits overnight before viewing as diffuse!

Quick PCR result

Thank you

• PCR experiments can be carried out easily

• Fun and relevant to wider world• Promote understanding