up 500 bp DNA of E.coli genome in deleting argR gene
When knocking out gene by using CRISPR-cas9 in E.coli, there should be a series of DNA that leads the genome repairing after cutting, so that the E.coli could live after knocking out a gene.
Usage and Biology
This sequence of DNA is used to help cas9 protein to cut the specific position on the DNA. It is constructed by connecting pUC, Ampr, RBS J23119, pTarget-upArm-gRNA-downarm. It is induced by the arabinose.
- Transform the plasmid pTarget with up-arm, down-arm, and gRNA to the competentE.coli DH10b from company transgene.
- Transform the plasmid pCas into the competent E.coli with previous plasmid mentioned.
- Add arabinose
- Cultivate the E.coli with the temperature at 30 degree Celsius overnight.
- Use colony PCR to test if the gene is successfully knocked out.
- As shown on the graph, the argR is knocked out with the decrease of 500bp which is the length of argR.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC