Hi guys,
I guess it is not that clever idea to try already cloned sequence into pDEST-EGFP-N1 vector to be “extracted” from the vector by restriction and inserted into pDEST-EGFP-C1 vector? Or I need to start from the pDONR vector?
I should admit, I haven’t looked carefully into the pDEST sequences….
Thank you in advance for your help!
Dear Marya
you can do it but not by using TOPO reaction.
You need to amplify the insert for first PDEST and insert in the second one.
You can do easily do it using PIPE cloning which require to perform 2 PCR (a PCR of the insert and a PCR of the vector missing of the region that you would like to replace with the insert (the ccdb - chloroanfenicol casssete in the case of pDEST)
pDEST e are an optimal vectors for the PIPE cloning because after the V_PCR you do not need to perform dpni digestion because the ccdb gene present into the vector ensure you that the vector used as template for the vector PCR will not give you colonies with empty pdest.
You can found more details about PIPE cloning and primer desing for it on my BLOG PRoteoCool ( https://proteocool.blogspot.com/ ) at page1: Gene cloning.
good luck
Manuele
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