Difference between revisions of "Part:BBa K3790161"
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The length of Sso7d-(G2S)3-Bst DNA was 1982 bp, which is approximately 2000 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions. | The length of Sso7d-(G2S)3-Bst DNA was 1982 bp, which is approximately 2000 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions. | ||
| − | [[File:T--Fudan--bst- | + | [[File:T--Fudan--bst-5.jpg|600px|thumb|none|'''Figure 1. PCR assembled Bst fusions.''' The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. The eighth lane (lane-5) was loaded with Sso7d-(G2S)3-Bst DNA.]] |
After ClonExpress homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight. | After ClonExpress homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight. | ||
Revision as of 19:40, 21 October 2021
Sso7d-(G2S)3-Bst
Contents
Introduction
The fusion protein obtained by linking the N-terminus of Bst to the C-terminal of Sso7d via a (G2S)3 linker.
Experimental Results
The length of Sso7d-(G2S)3-Bst DNA was 1982 bp, which is approximately 2000 bp after adding homology arms to both ends for PCR cloning. We isolated the target DNA by gel extraction for subsequent reactions.
After ClonExpress homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with antibiotics. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with antibiotics, 37℃ shaking overnight.
Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]