Transcript DNA Fingerprinting

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/c
hemistry/laureates/1993/mullislecture.html
What is in a PCR reaction
Use 5μl from tube labelled
“Template DNA”
Template DNA
The starting material in a
PCR reaction.
Use 20μl from tube
labelled “primers”
Primers are two short
pieces of DNA (0-15
bases long) that
determine the region of
DNA to be copied.
The PCR bead
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
Mix the following:
•
•
•
•
How does PCR
work?
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!
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
B
C
M LG1 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