Lab Protocols

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• pDECKO cloning protocol. Here a detailed protocol for pDECKO plasmids cloning
• Design spreadsheet. Here a spreadsheet to help with the design of the 6 oligos corresponding to the Insert-1 in the pDECKO cloning

Overview of DECKO Cloning

This document describes the cloning of individual targeting plasmids (as opposed to library cloning). DECKO employs a 2-step cloning methodology. Cloning is based on the Gibson assembly method, which avoids conventional ligation and enables multi-fragment assembly. In Step 1, multiple oligonucleotides are assembled together with the Backbone plasmid to create an intermediate plasmid. In Step 2, a constant "Insert-2" fragment is inserted within the new sequence created in the previous step. Insert-2 sequence can be amplified by PCR using oligos and Template plasmid described below. The time required for the complete cloning is about 5 days.

Fig. 1. Diagram showing: (A) final pDECKO_mCherry construct (B) step1 cloning (multiple oligo assembly) (C) step 2 cloning (constant fragment assembly)

Fig. 2. Detail of 6 oligo assembly example.