BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
Protocols of Nucleic Acid Detection for Molecular Diagnosis of
I. Primer and Probe Reconstitution
A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are
required because the enzymes that catalyze replication, DNA polymerases, can only add new
nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the
primer, and copies the opposite strand. The length of a primer is usually between 18 – 30 bases.
Primer Sequences :
1.1. Reagents
- Primer or probe
- Nuclease Free Water (NFW)
1.2. Equipments
- Vortex mixer
- Microtube 1.5 mL
- Micropipette 1000 µL
- Micropipette 100 µL
- Micropipette 10 µL
- Aerosol barrier pipette tips 100 – 1000 µL
- Aerosol barrier pipette tips 10 – 100 µL
- Aerosol barrier pipette tips 1 – 10 µL
1.3. Procedures
1. During the reconstitution process, primer and probe should be placed on the ice or ice
box.
2. Probe is light sensitive, so protect them from light and work fast.
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
3. Make primer stock solution with 100 µM concentration by adding X ml of NFW. By
definition a 100 µM solution would be 100 µmol / L.
Primer and probe equation for reconstitution can be done by using examples below:
a. Reconstitution based on molecule quantity.
For example, the quantity of a primer is 51.2 nmol.
51.2 nmol ∞ 0.0512 µmol
Then, solve the following equation for X to identify the volume needed to achieve
the working concentration.
0.0512 µmol = 100 µmol
X
L
By cross-multiplying, X = 0.0512 µmol x L
100 µmol
X = 0.000512 L ∞ 512 µl
b. Reconstitution based on molecular weight (MW) and weight.
For example, the MW of a primer is 6115.9 µg/µmol, and the weight is 313.3 µg
First convert the µg to µmol using this formula :
µmol = µg
MW
= 313.3 µg
6115.9 µg/µmol
= 0.0512
To make primer concentration to 100 µM :
100 µM = µmol
X
X = 0.0512
100
X = 0.000512 L ∞ 512 µL
4. Based on the examples above, in order to make 100 µM, add nuclease free water 512
µl. Vortex the tube to mix them.
5. Now we have primer with 100 µM concentration, this will be our stock solution. If for
this primer, we will use 5 µM for one reaction. In order to make ready to use primer
with 5 µM concentration with 100 µL volume, we can use this formula:
V1 x M1 = V2 x M2
Where : M1 = Concentration of the stock solution
M2 = Concentration of the desired primer
V2 = Volume of the desired primer
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
V1 = Volume of the primer which we have to take from the stock
solution primer
V1 x 100 µM = 100 µL x 5 µM
V1 = 5 µL
6. Based on the above equation, take 5 µL of primer from stock solution, put in a new 1.5
mL microtube, then add 95 µL of nuclease free water. Vortex the microtube to mix
them. Now we have ready to use 100 µl of primer with concentration 5 µM.
7. Label each micotubes by putting the name of the primer, date of dilution and
concentration of the primer and probe.
8. Store diluted primer and probe in -20o freezer.
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
II. Fast SYBR Green® Master Mix Optimization Protocol (2 Steps RT-PCR)
Fast SYBR Green Master Mix is designed to deliver PCR results on Fast instruments in less than
half the time of standard real-time PCR reagents while maintaining the equivalent PCR
performance on Fast PCR. This master mix was formulated to minimize primer dimerization and
non-specific amplification to optimize the efficiency and accuracy of the reaction without
compromising sensitivity. This master mix also allows the user to do the real-time PCR test
without using probe.
2.1. Reagents
- Fast SYBR Green® Master Mix 1-Pack (Applied Biosystems cat. no : 4385612)
- Nuclease Free Water
- Primers
- cDNA
2.2. Equipments
- Vortex mixer
- Microtube 1.5 mL
- Micropipette 1000 µL
- Micropipette 100 µL
- Micropipette 20 µL
- Micropipette 10 µL
- Aerosol barrier pipette tips 100 – 1000 µL
- Aerosol barrier pipette tips 10 – 100 µL
- Aerosol barrier pipette tips 2 – 20 µL
- Aerosol barrier pipette tips 1 – 10 µL
- MicroAmp® Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL (Applied
Biosystems cat. no: 4346906)
- MicroAmp® Optical Adhesive Film (Applied Biosystems cat. no: 4311971)
- MicroAmp® Adhesive Film Applicator (Applied Biosystems cat. no: 4333183)
- MicroAmp® 96-Well Support Base (Applied Biosystems cat. no: 4379590)
2.3. Procedure
1. Pipetting is crucial in fast real time PCR. Take care to not carryover extra reagents or
sample volumes by putting pipette tip too far into containers or resting the body of the
tip on the side of container.
2. Always wear powder-free gloves when preparing the reaction mix for real-time PCR test.
3. Prepare a 96-well reaction plate as shown in Table 1.
• Use 10 to 100 ng of genomic DNA or 1 to 10 ng of cDNAtemplate.
• The final concentration of Fast SYBR Green Master Mix is 1✕.
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
4. The plate configuration accounts for two replicatesof each of the following nine variations
of primer concentration applied to both template and NTC wells:
Reverse Primer
(nM)
50
300
900
50
50/50
50/300
50/900
Forward Primer (nM)
300
300/50
300/300
300/900
900
900/50
900/300
900/900
5. Put the plate sitting on top of the support base.
6. Load the primers and nuclease free water into the appropriate wells in an MicroAmp®
Fast Optical 96-Well Reaction Plate, according to Table 1 .
7. Carefully dispense 2 μL cDNA into each test well.
8. Seal the plate with a MicroAmp® Optical Adhesive Film.
9. Optional: Spin the plate briefly in benchtop centrifuge with 96-well plate adaptors, to
collect the reaction mix at the bottom of the well.
10. Program the thermal-cycling conditions and run the samples according to the Appendix
1.
11. If the plate is not going to be run immediately, it can be stored in the fridge 4o C for about
6 hours prior running it by real-time PCR machine.
12. Compile the results for Rn and CT, then select the minimum forward and reverse primer
concentrations that yield the maximum Rn values and low CT values.
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
Appendix 2
SYBR Green Real Time PCR Worksheet Using INFA CDC Primer (2 Steps RT-PCR)
Date :
Operator :
Test :
Reaction Set Up
Reagent
Amount per-reaction
Primer Forward ( 5 µM)
Primer Reverse ( 5 µM)
Fast SYBR® Green Master
Mix
variable µL
variable µL
10 µL
Nuclease Free Water
Mix volume
variable µL
18 µL
cDNA template
2
Total volume
20 µL
Total Number of
Reactions
Total Amount of Reagent
Dispense 18 µL into wells
of optical plate
Add 2 µL of cDNA
template into each wells
µL
PCR Plate Assay Layout Sheet
1
2
3
4
5
6
A
B
C
D
E
F
G
H
Cycling Conditions :
Stage 1 : 95.0o C
20 seconds
Stage 2 : 95.0o C
60.0o C
3 seconds
30 seconds
no repeat
40 repeats
7
8
9
10
11
12
BSL-X XXXX Standard Operating Procedures V3-XXX
Author: Maureen T. Long DVM, PhD
Infectious Diseases & Pathology
College of Veterinary Medicine
Review Date: 2/9/2016
Signature:
Table 1 :
Plate Configuration of Primer Optimization for PCR
Wells
A1 - A2
A3 - A4
A5 - A6
A7 - A8
A9 - A10
A11 - A12
B1 - B2
B3 - B4
B5 - B6
C1 - C2
C3 - C4
C5 - C6
C7 - C8
C9 - C10
C11 - C12
D1 - D2
D3 - D4
D5 - D6
Fast SYBR
Green
Master Mix
(μL)
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Master Mix
5 μM
Forward
Primer
(μL)
0.2
0.2
0.2
1.2
1.2
1.2
3.6
3.6
3.6
0.2
0.2
0.2
1.2
1.2
1.2
3.6
3.6
3.6
Fwd Primer
A1-A6,
C1-C6
130
2.6
A7-A12,
C7-C12
130
15.6
B1-B6,
D1-D6
130
46.8
5 μM
Reverse
Primer
(μL)
0.2
1.2
3.6
0.2
1.2
3.6
0.2
1.2
3.6
0.2
1.2
3.6
0.2
1.2
3.6
0.2
1.2
3.6
Template
(μL)
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
Deionized Total
Water
Volume
(μL)
(μL)
7.6
6.6
4.2
6.6
5.6
3.2
4.2
3.2
0.8
9.6
8.6
6.2
8.6
7.6
5.2
6.2
5.2
2.8
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20