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Team:TU Darmstadt/Notebook/Labjournal/K1497032
From 2014.igem.org
Scaffold-His-Cys
A mutagenesis PCR was used to insert a cysteine residue at the C-terminal part of the scaffold His-tag protein. The correct size of the PCR product was checked via gel electrophoresis. In figure 7 a strong band is visible in the expected height of 6234 bp. The smear above might be the result of an overloaded lane.
The PCR product was self-ligated and E. coli Top 10 cells were transformed with the construct. The PCR product was phosphorylated prior ligation and 2 µL T4 DNA ligase and an incubation time of 2 h were used, additionally. The procedure was adjusted to an online protocol [bitesizebio.com/19149/7-ways-to-improve-blunt-end-ligations (23.07.14)] in order to increase the blunt end ligation efficiency. In the succeeding colony PCR, all five clones showed a positive result of bands migrating at the expected height of 943 bp (see fig 1). Moreover, sequencing of the extracted vectors verified the correct insertion of the cysteine residue in all three chosen clones.
Figure 1: Mutagenesis PCR of the scaffold His-tag protein (MutPCR) for the insertion of a cysteine at its C-terminus. On the left side the 2-log ladder (M) was applied. The PCR product migrates at the expected height of 6234 bp.
Figure 2: Colony PCR of the mutagenesis PCR. The 2-log ladder (M) was applied onto the left lane. The amplicon (SHC) has a length of 943 bp. The amplificate from 4.2 was applied onto the right lane as a positive control.
