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218 Kohlmann et al.
sis procedure and maximizes recovery of cDNA. Synthesis for the second
strand takes place using the DNA/RNA hybrid as substrate. Mild treatment
with RNase H inserts nicks into the RNA, providing 3� OH-primers for DNA
polymerase I present in the second-strand enzyme cocktail. The 5�-3� exonuclease
activity of DNA polymerase I removes the primer stretches in the direction
of synthesis, which are then replaced with new nucleotides by the
polymerase activity. Escherichia Coli ligase links the gaps to a complete ds
cDNA strand. The last step in the cDNA synthesis is to ensure that the termini
of the cDNA are blunt. This is done by adding T4 DNA polymerase, which
removes any remaining overhanging 3� ends on the ds cDNAs.
1. Thaw all necessary components and place them on ice. Pipet the following components
in a sterile 1.5-mL reaction tube (40 µL total reverse-transcription reaction
volume) (see Table 1).
2. Incubate 10 min at 70°C (Eppendorf Thermostat Plus; can also be used for all
following downstream incubations), then place the tube immediately on ice. Add
the components from Table 2, mix gently, and incubate for 60 min at 42°C. In
the meantime, thaw all required components for the second-strand synthesis
reaction, mix them, and place on ice.
3. After 60 min, place the tube on ice for 5 min to terminate the reaction. Continue
immediately with the second-strand reaction (see Table 3). Pipet directly into the
first-strand reaction tube the following components, mix gently, and incubate for
2 h at 16°C.
4. After 2 h incubation, add 20 µL (20 U) T4 DNA polymerase and incubate for 5 min
at 16°C. Then stop the reaction by adding 6.8 µL EDTA (0.5 M, pH 8.0).
5. Subsequently, digest residual total RNA. Add 1.5 µL (15 U) RNase I and incubate
for 30 min at 37°C. Add 5 µL (3 U) proteinase K to the reaction and incubate
for another 30 min at 37°C.
6. Add 153.5 µL water to the cDNA. The final volume now is 336.8 µL and the
cDNA is ready for the subsequent cleanup step.
3.3.3. Cleanup of ds cDNA
The cDNA cleanup step is performed using 1.5-mL Phase Lock Gel (PLG)
technology caps (Eppendorf). PLG is a product that eliminates interface-protein
contamination during the phenol extraction. Upon centrifugation, the gel
migrates to form a tight seal between the phases of an aqueous/organic extraction.
The organic phase and the interface material are effectively trapped in or
below the barrier. This allows the complete and easy transfer of the entire aqueous
phase containing the cDNA species by simply pipetting. The risk of contaminating
the sample with interface material is eliminated.
1. Add 330 µL phenol/chloroform/isoamylalcohol (25:24:1) to the cDNA solution,
vortex for 10 s, and transfer the supernatant to a 1.5-mL PLG tube. Centrifuge for