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Class of Modern Life Science Laboratory
- May, 2014 -
T.A Hong Nhung Nguyen & 서보경
Experiment 10
Polymerase Chain Reaction (PCR)
Contents
①Introduction
②Materials and Methods
③Result and Discussion
④Trouble shooting
Purpose of today’s experiment
What is PCR?
Purpose of today’s experiment① Introduction
www.expeditions.udel.edu
Our previous experiments
(A)
(B)
(C)
Purpose: Amplify a “DNA fragment of interest” in genomic DNA, total cDNA, plasmid.
Dr. Kary Mullis, the inventor of PCR, was
awarded the 1993 Nobel Prize in Chemistry.
PCR invention and definition① Introduction
PCR is a technique which is used to
amplify a specific region of DNA, in order
to produce enough DNA to be adequately
tested.
A special DNA polymerase (Taq) is used to
make many copies of a short DNA fragment
(100-10,000 bp) defined by primers.
81,267,844 bp 81,572,676 bp
start end
ATG10 is located at 5q14.1, size: 304,833 bases
Autophagy Related Gene 10 (ATG10) in Chromosome 5
ATG10
….PCR to amplify a DNA
fragment of interest (861 bp)
① Introduction
81,267,844 bp 81,572,676 bp
start end
ATG10 is located at 5q14.1, size: 304,833 bases
ATG10
….PCR to amplify a DNA
fragment of interest (861 bp)
Forward and Reverse primers location in genomic DNA
for PCR (to amplify a “DNA fragment of interest)Materials and Methods
1. DNA template
2. Primer (10 pmol)
3. Taq DNA Polymerase
4. dNTPs mixture (10 mM)
5. Taq Buffer 10X and 25 mM MgCl2 mixed
6. 5X band doctor (Purchased from Solgent) use only when template DNA is genomic DNA
PCR machine
1. DNA (genomic DNA isolated from human or Arabidopsis) Stock concentration: 500 ng/ul
2. cDNA (complementary DNA) synthesize from RNA after Reverse Transcriptase (RT)
3. Plasmid DNA Stock concentration: 50 ug/ul
Mixture of Forward and Reverse primer (Listed as detail in your protocol)
Stock 5U/ul, speed of synthesize: 1 minute/1 kb
Mixture
Add DNA template to each sample
Experimental design and prepare the mixture
PCR machine programming
PCR machine
Thermocycles
Methods Programming Polymerase Chain
Reaction
GeneSequence
(5’-->3’ direction)Template DNA Tm
Expected size(kb)
gDNA
ATG10-F GAA CAT CCA ATA CTT GGG CAA C
MCF-7 gDNA 0.86ATG10-R AGG GAC ATT TCG TTC ATC CTG
53
53? Extension time
PCR in test tubes
Melting
94 oC
Tem
per
ature
100
0
50
Time
5’3’
3’5’
PCRMelting
94 oC
Tem
per
ature
100
0
50
Time
3’5’
5’3’
Heat
PCRMelting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
3’5’
5’3’5’
5’
Melting
94 oC
PCRMelting
94 oC
Melting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
PCRMelting
94 oC
Melting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
PCRMelting
94 oC
Melting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
Heat
Heat
PCRMelting
94 oC
Melting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
5’
5’
5’
5’
Fragments of defined length
PCRMelting
94 oC
Melting
94 oCAnnealing
Primers
50 oC
Extension
72 oC
Tem
per
ature
100
0
50
Time
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
5’
5’
5’
5’
Why PCR product using cDNA as template sometimes smaller in size?
Result
PCR using ATG10 F/R specific primer with different templates
Where primer should be pick to check mRNA level?
861 bp
207 bp
GeneSequence
(5’-->3’ direction)Template DNA
Annealing
Tm
Expected size(kb)
gDNA
ATG10-F GAA CAT CCA ATA CTT GGG CAA C
MCF-7 gDNA 0.86ATG10-R AGG GAC ATT TCG TTC ATC CTG
53
53
http://genome.ucsc.edu
Isoform 1
Isoform 2
Isoform 3
861 bp
207 bp
Expected size
Common troubles in PCRTroubleshooting
Contamination??
Expected size
No band??
??
Summary 1. PCR is a technique which is used to amplify a specific
region of DNA, in order to produce enough DNA to be
adequately tested.
2. PCR is very sensitive
3. PCR need to be well-design and performin optimal condition
Today: Amplify a “DNA fragment of interest” in
genomic DNA, total cDNA, plasmid.
861 bp
207 bp
