09 September 2019 3 7K Report

Hello everyone

I would like to ask your help in understanding some point,

I am trying to clone a very long gene around 4000bp , I used the method of HiFi assembly Kit which require designing primers with an overlaps between them to make four fragments of this gene and then use PCR reaction to amplify them by the KOD plus kit. I succeed in the amplification reaction , and now time to incubate the vector (pCDNA3)with the RE (BamH1, EcoR1), cut smart buffer and water for overnight at 37c , but in my ligation reaction I add the vector after cutting with the insert though I don't use RE with the insert . do i need to ligate my insert fragments together and then added them to my vector to ligate them all

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