13611
doi
10.5281/zenodo.13611
oai:zenodo.org:13611
Transformation of plasmid DNA to competent E. Coli cells
sprotocols
ScientificProtocols.org
info:eu-repo/semantics/openAccess
Creative Commons Zero v1.0 Universal
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Authors: Chenzhong Kuang
### Material and Reagents
1. SOC
- 2% Tryptone
- 0.5% Yeast Extract
- 10mM NaCl
- 10mM MgSO4
- 10mM MgCl2
- 1.5 mL microfuge tubes
- 42° C waterbath
- Ice
- 37° C shaker
### Protocol
1. Thaw competent cells on ice. 20–200µL per tube
- Add max. 20µL of a ligation reaction
- Mix very gently!
- Incubate the tubes on ice for 30 min
- Heat shock the cells for 45 sec to 2 min at 42°C
- Place the tubes immediately on ice for at least 2 min
- Add 800µL of SOC medium to each tube
- Incubate for 1 hour at 37°C and shake vigorously
- Spin down briefly and remove most supernatants
- Resuspend cell pellet with the rest SOC medium in the tube by pipetting
- Plate out the suspension on a LB agar plate containing the appropriate antibiotic.
- Incubate the plates overnight at 37°C
### Remarks
1. I have use this protocol to transform DH5 alpha competent cells successfully
- When transforming purified plasmid into competent cells, I just add 1µL plasmid DNA solution. Then plate out only 10–20µL bacterial suspension to the plate instead of all
- Efficiency depends on ligation reaction and competent activity.
Zenodo
2014-12-30
info:eu-repo/semantics/article
607024
1579530712.504455
1168
md5:1d34366902ec0698ba2b5155d1b72205
https://zenodo.org/records/13611/files/protocol.md
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