Beruflich Dokumente
Kultur Dokumente
(PCR)
Use 5l from tube labelled Use 20l from tube labelled The PCR bead
Template DNA primers
MgCl2
Required for Taq DNA
polymerase to function
BAGAIMANA PCR BEKERJA?
Template DNA
Nucleotides
Primers
Taq DNA polymerase
MgCl2 ++ ++ ++
JUMLAH DNA YANG DIPRODUKSI
Forensic medicine.
Preimplantation Genetic Diagnosis (PGD).
Arkeologi.
Paternity testing.
FORENSICS!
PENGGUNAAN PCR UNTUK
FORENSIK
PCR bisa digunakan untuk mengamplifikasi DNA dari
sejumlah kecil sel (sekitar 1000 sel).
DNA yang telah diamplifikasi bisa digunakan untuk
menganalisis DNA fingerprinting untuk menentukan
tersangka pelaku kejahatan.
DNA FINGERPRINTING MENGGUNAKAN
PCR DALAM INVESTIGASI FORENSIK
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
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
50
0
Time
3 5
5 3
Temperature
100
Melting
94oC
50
0
Time
3 5
Heat
5 3
Temperature
100
Melting Melting
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
Time
3 5
5
5
5 3
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
Time
3 5
Heat
5
Heat
5
5 3
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
Time
3 5
5
5
5
5
5
5
5 3
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
3 5 5 Time
5
5
5 3
Heat
5
Heat
5
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
3 5 5 Time
5
5 5
5 3
5
5
5
5
5
5
Temperature
100
Melting Melting 30x
94oC Extension 94oC
Annealing
Primers 72oC
50 50oC
0
3 5 5 Time
5
5 5
5 3
5
Fragmentsof 5
definedlength
5
5
5
5
DNA
ELECTROPHORESIS
GEL CASTING
RUNNING BUFFER
TAE buffer
20ml 50x buffer to 1 litre with water
A B C D E F
M LG1 LG2
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