Islamic University of Jerusalem
Real Time PCR Training Course
Working Plan
Dr. Basim Ayesh
August,
2009
1
Objectives:

Design and apply a real-time PCR experiment using the SYBR green dye method.

Learn to use the SDS software for the 7500 real time PCR system from Applied Biosystems.

Apply a standard curve quantitation method using the SYBR-green dye.

Examine different representations of results analysis, and examine the results of changing different parameters (ie.
Threshold and baseline) on the Ct value and quantitation results.

Examine the effect of variable Primers concentrations on the quality of SYBR-Green PCR amplification of 5 different targets.

Distinguish between specific and non specific amplification products using the melting curve method.

Determine whether the 5 different targets can be multiplexed in a single tube based on the melting points analysis.
2
PCR plate/strips Setup
A
B
C
D
E
F
1
2
3
4
5
6
7
8
1 x 106
1 x 106
1 x 106
U
U
U
U
U
1 x 105
1 x 105
1 x 105
DW
DW
DW
DW
DW
1 x 104
1 x 104
1 x 104
U
U
U
U
U
1 x 103
1 x 103
1 x 103
DW
DW
DW
DW
DW
1 x 102
1 x 102
1 x 102
U
U
U
U
U
1 x 101
1 x 101
1 x 101
DW
DW
DW
DW
DW
9
10
11
12
G
H
Primer concentration: 0.1μM each
U = unknown patient sample
Primer concentration: 0.2μM each
DW = PCR-grade double distilled water
Primer concentration: 0.3μM each
3
Working Sheet
Operator
Names
Column
Target
MMX
1, 2, 3
PBGD
A, B, C
Group 1
4
PBGD
A, B, C
Group 2
5
MRP
A, B, C
Group 3
6
LRP
A, B, C
Group 4
7
BCRP
A, B, C
Group 5
8
MDR-1
A, B, C
Instructor
Single-Tube Reaction Mixtures
Reagent
Mixture A
Mixture B
Mixture C
2X SYBR-Green MMX
10 μl
10 μl
10 μl
Forward primer (2μM)
1 μl
2 μl
3 μl
Forward primer (2μM)
1 μl
2 μl
3 μl
DW
3 μl
1 μl
0 μl
cDNA (about 20ng/μl)
5 μl
5 μl
5 μl
Total
20 μl
20 μl
20 μl
4
Trainees Working Sheet
Group Number: …………………………
Samples:
1. ………………………………………............
Reagent
2. …………………………………………………
2X SYBR-Green MMX
3. …………………………………………………
4. …………………………………………………
Mixture A
Mixture B
Mixture C
Forward primer (2μM)
Forward primer (2μM)
DW
5. …………………………………………………
cDNA (about 20ng/μl)
6. …………………………………………………
Total
5