The first step was to clone individual sgRNA into these entry vectors by BpiI digestion and ligation.
We generated entry vectors (pEV) used for the first step of the Golden Gate cloning method by inverse PCR using a Platinum Gate TALEN kit as a template42.
To generate pCRC::YUC4, the 3.5 kbp CRC promoter and YUC4 coding sequence were amplified using genomic DNA and cDNA from Ler as PCR templates and the products were sub-cloned into different entry vectors.
c and d Target cloning to the entry vectors.
The two entry vectors should be used in turns when targeting multiple sites.
The genome editing tool suites contain two additional compatible entry vectors, pEntry A and pEntry B, for packing the sgRNA cassette (Fig. 1j).
Resulting entry vectors were LR recombined with pYES-DEST52.
All Entry vectors were propagated in the E.coli strain TOP10F' (Invitrogen) under kanamycin selection.
cDNAs, shRNAs or miRNAs are initially cloned into Entry vectors (Figure 1).
The coding regions of MKK4 on all the Entry vectors were verified by DNA sequencing.
With a wide variety of destination vectors, ORFs cloned into the entry vectors can be rapidly transferred for different applications.
