Should I amplify my eBlocks Gene Fragments when I receive them?

eBlocks Gene Fragments are synthesized from several smaller overlapping fragments. Even after purification, the final prep will contain some of these smaller byproducts. During PCR smaller products are more efficiently amplified than longer ones, and once the exponential phase of PCR is reached, any smaller products in the reaction will overwhelm the generation of the full-length correct product. Researchers will often see a smear or smaller bands than expected when the PCR product is run on a gel.

Most applications, such as restriction digest, blunt end, and Gibson cloning, can be performed with the material provided during the original synthesis, and PCR amplification should not be necessary. If the purpose of the amplification is to add flanking bases required to complete a cloning reaction, we strongly recommend ordering the eBlocks fragment with the required bases rather than adding them afterwards via PCR.

If you must PCR amplify your eBlocks fragments, use the minimum amount of starting template possible according to your PCR kit protocol, limit the cycle number to 10–12, and use a high-fidelity polymerase.