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Transforming into DH5α or TOP10 E. coli
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When transforming into DH5-alpha E.coli cells we followed the protocol below: | When transforming into DH5-alpha E.coli cells we followed the protocol below: | ||
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| - | * On ice, add 12.5µl of the ligation mixture (see [[Ligating DNA]]protocol to 100µl of DH5-alpha E.coli cells in an eppendorf tube | + | * On ice, add 12.5µl of the ligation mixture (see [[Ligating DNA]] protocol) to 100µl of DH5-alpha E.coli cells in an eppendorf tube |
* Mix well by pipetting up and down several times | * Mix well by pipetting up and down several times | ||
* Leave on ice for 45 minutes | * Leave on ice for 45 minutes | ||
Latest revision as of 16:06, 28 October 2008
Newcastle University
GOLD MEDAL WINNER 2008
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Home >> Wet Lab >> Protocols >> Transforming into DH5α or TOP10 E. coli
When transforming into DH5-alpha E.coli cells we followed the protocol below:
The plasmids we transformed were present in a 25µl ligation mixture (6µl of this being the vector plasmid, and 15µl of this being the digested plasmid in which the desired 2.2kb fragment was restricted out of).
- On ice, add 12.5µl of the ligation mixture (see Ligating DNA protocol) to 100µl of DH5-alpha E.coli cells in an eppendorf tube
- Mix well by pipetting up and down several times
- Leave on ice for 45 minutes
- Remove from ice and incubate for 3 minutes at 37˚C (this is the heat shock)
- Add 300µl of LB and mix well with pipette
- Incubate for 1 hour in a 37˚C water bath
- Plate onto antibiotic agar (see Making Agar Plates )
