3.4. cDNA purification

Although purification of cDNA is optional, it is highly recommended to include this step. It is designed for rapid purification of single-stranded or double-stranded PCR amplification products (i.e. 100 base pairs to 10 kilobase) from the other components in the reactions, such as excess primers, nucleotides, DNA polymerase, oil and salts. Different cDNA purification kits are commercially available (e.g. QIAquick PCR Purification Kit^® from Qiagen, DNAclear™ kit from Applied Biosystems, Jetquick PCR Purification Kit from Genomed). The protocol here below is outlined using the GenElute™ PCR Clean-Up Kit (Sigma, USA).

Insert a GenElute™ plasmid mini spin column, with a blue o-ring, into a provided collection tube if not already assembled.

Add 0.5 mL of column preparation solution to each GenElute™ plasmid mini spin column.

Centrifuge at 12000xg for 30 s to 1 min.

Discard the eluate.

Add 5 volumes of binding solution to 1 volume of the PCR reaction and mix (i.e. add 500 µL of binding solution to 100 µL of the PCR reaction).

Transfer the solution into the binding column.

Centrifuge the column at 12000xg to 16000xg for 1 min.

Retain the collection tube and discard the eluate.

Replace the binding column into the collection tube.

Apply 0.5 mL of diluted ethanol-containing wash solution to the column.

Centrifuge at 12000xg to 16000xg for 1 min.

Retain the collection tube and discard the eluate.

Replace the binding column into the collection tube.

Centrifuge at 12000xg to 16000xg for 2 min without any additional wash solution.

Discard any residual eluate as well as the collection tube.

Transfer the binding column to a fresh 2.0 mL collection tube.

Apply 50 µL of elution solution or water (see Note 9) to the center of each column.

Incubate at room temperature for 1 min.

Centrifuge the binding column at 12000xg to 16000xg for 1 min.

The PCR amplification product is now present in the eluate and is ready for immediate use or storage at -20°C.