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Team:TU Darmstadt/Notebook/Labjournal/K1497027
From 2014.igem.org
Construction of scaffold double domain vectors in pSB1C3
Two single domains of the scaffold protein were joined together by inserting a single domain into the earlier constructed single domain vectors. The backbone vectors were digested with BamHI and PstI. The inserts were amplified with primer containing the BglII, EcoRI, and XbaI restriction site and the BamHI, SpeI, and PstI hestrictions site. The backbones were ligated with the inserts and Top10 Zells were transformed with the products. The colonies were screent via colonie PCR and positive colones were grown in LB-Cmp-Medium. The plasmids were recoverd via Mini-Plasmid-Preparation.
Figure 1: Backbone production for scaffold double domain vectors.
The plasmids pSB1C3-GBD , pSB1C3-PDZ, and pSB1C3-SH3 were digested with BamHI and PstI. Only names of the respective domains are shown. A sample of the undigested vector (left) was loaded next to the digested vectors (right). The digested vectors migrate at their expected heights, as well as the initial vectors. The 2-log marker (M) is shown on the left.
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Figure 2: Colony PCRs after transformation of E. coli Top10 cells with the ligation relations.
Triplicates of the PCR reactions for the obtained double domains are shown. The 2-log marker (M) is shown on the left. Kolonie 2 (middle) was always positive for all constructs.
