BBa_K929300
BBa_K929300 Version 1 Component
This part has been discontinued.
Please consider using BBa_K929301 as a replacement.
Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: iGEM Potsdam Bioware 2012
Date created: 2012-09-23 11:00:00
Date modified: 2015-05-08 01:13:47
Potsdam Standard Backbone
| Types | DnaRegion |
| Roles | plasmid_vector Plasmid_Backbone |
| Sequences | BBa_K929300_sequence (Version 1) |
Description
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method (Blanusa et al. (2010)). For this standard we designed a new cloning backbone with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3, the Potsdam Standard Backbone. In the prefix, we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3??? overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that???s the only step where we have to use restriction enzymes.The gene of interest was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5??? end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3??? overhang which is suitable to the overhangs that was created by cutting the new assembly vector.
After that the digested vector and the PLICed insert were mixed and transformed into E. coli. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.