Is it necessary to gel extract either the vector or the PCR insert prior to the in-fusion HD cloning reaction? (as suggested by Takara)
I believe they recommend to gel-purify PCR insert not the vector.
However, if your PCR reaction shows a strong single band on the gel, I wouldn't bother to gel-purify it.
Hi Cindy,
I used to do gel purification of both digested vector as well as PCR amplified insert for in-fusion HD cloning reaction.
Thank you! I understand how one can get away with only a spin column purification of the PCR insert when there is only one band on the gel. Can one just do a spin column purification for the linearized vector?
For Fusion-HD, I usually linearize the vector with two enzymes so that the cutting efficiency is high.
Therefore, I do not gel-purify a linearized vector.
However, one could gel-purify the linearized vector if a single enzyme was used to linearize.
I usually do a gel extraction and purification for digested vector as well as PCR amplified insert for in-fusion HD cloning reaction to insure the removing of any digestive enzyme or uncut vector from the reaction .
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