III. Second strand Synthesis and amplification
Optimisation of LD-PCR second strand synthesis / cDNA amplification (SMART
PCR cDNA Synthesis Kit protocol p19)
Follow the protocol outlined in section VII.B of the Clontech SMART cDNA synthesis
kit manual, with some modifications:
1. Preheat a thermal cycler to 95°C.
2. Prepare a Master Mix by combining the following components in the order
shown. Multiply the volume of each by the number of samples:
18.5 μl
Deionized H2O
2.5 μl
10X Advantage 2 PCR Buffer
0.5 μl
50X dNTP Mix (10 mM of each dNTP)
0.5 μl
5' PCR Primer II A (12 μM)
0.5 μl
50X Advantage 2 Polymerase Mix
22.5 μl
Total volume
3. Mix well by vortexing and centrifuge the tube briefly in a microcentrifuge.
4. Aliquot 22.5 μl of the Master Mix into each reaction tube.
5. Add 2.5 µl of the first strand reaction
6. Mix contents by gently flicking the tubes. Centrifuge tubes briefly in a
microcentrifuge.
7. Cap the tube, and place it in the preheated thermal cycler. Run the following
program: 95°C 1 min, followed by 27 cycles of 95°C 15 sec, 65°C 30 sec, 68°C 6
min. Take a 5 µl sample after 15, 18, 21 and 24 cycles and check it on a 1.8%
agarose/EtBr gel (0.5X TAE buffer). 5 µl is enough if for the gel if you use very
narrow wells. Pause the PCR program to take the sample.
8. The amount of cDNA should reach a plateau; select the lowest number of cycles
necessary to reach the plateau.
Preparative LD-PCR second strand synthesis / cDNA amplification (SMART PCR
cDNA Synthesis Kit protocol p19)
1. Preheat a thermal cycler to 95°C.
2. Prepare a Master Mix by combining the following components in the order
shown. Multiply the volume of each by the number of samples and the number of
amplifications required per sample (usually 2 or 3 100µl reactions are enough to
produce 4 µg of cDNA):






74 μl
10 μl
2 μl
2 μl
2 μl
90 μl
Deionized H2O
10X Advantage 2 PCR Buffer
50X dNTP Mix (10 mM of each dNTP)
5' PCR Primer II A (12 μM)
50X Advantage 2 Polymerase Mix
Total volume
Note: 1µl of 50X Advantage 2 Polymerase Mix per 100µl gives as much product as
2µl (Mark Cock, Personal communictiaon) so it should be possible to use less
enzyme.
3.
4.
5.
6.
Mix well by vortexing and centrifuge the tube briefly in a microcentrifuge.
Aliquot 90 μl of the Master Mix into each reaction tube.
Add 10 µl of the first strand reaction
Mix contents by gently flicking the tubes. Centrifuge tubes briefly in a
microcentrifuge.
7. Cap the tubes, and place in the preheated thermal cycler. Run the following
program: 95°C 1 min, followed by the optimum number of cycles (determined
above) of 95°C 15 sec, 65°C 30 sec, 68°C 6 min.