Frequently asked questions

Our Scientific Applications Support team has assembled a list of frequently asked questions to help you find answers quickly. Filter using one or more categories to focus on specific topics, or use the search bar to perform a text search.

How can I use PCR to add overlapping regions to my gBlocks™ Gene Fragments that I will use in a Gibson Assembly reaction?

Don’t use PCR to add the overhangs, because PCR can preferentially amplify smaller byproducts in the prep, leading to issues with the cloning reaction and increasing the number of colonies you need to screen to find the correct full-length insert.

Instead, build the overhangs into the gBlocks fragment itself. The amount of gBlocks fragments you will receive is more than enough for a couple of assembly reactions, so the additional PCR step is not required to generate additional material and just adds an additional enzymatic step to the protocol that can introduce errors.



Tags:
iGEM