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? “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

Mullis’s Nobel prize speech is well worth reading http://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. 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

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

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! ... Like TV shows...

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 seconds 71°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 start Programme 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 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

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