IGEM:MIT/2007/Notebook/2007-6-14
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Grad Advisors
- Morning: Debbie, Forrest
- Afternoon: Brian
LAB WORK
- Make competent E. coli cells
- Transformation into competent cells
- Plate transformed cells
Depending on how fast cells grow......
- Night time
- Analyze plates
- innoculate 4ml LB/Amp
Procedures
Regular cell membranes are not permeable to plasmids, so we much use ions (CaCl) to make the bacteria competent and able to accept foreign DNA.
Start: 2 vials of competent bacteria (500 µL each)
9 Transformations
Contents Volume Label U V (2 µL) I dV (5 µL) A dV + dI (5 µL) B ddV (5 µL) C ddV + ddI (5 µL) D dV (10 µL) E dV + dI (10 µL) F ddV (10 µL) G ddV + ddI (10 µL) H
Transformation Protocol
- Aliquot 5 µL and 10 µL of ligation reactions into eppendorfs
- For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/µL)
- Pipette 50 µL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
- Do not pipette up and down
- Incubate on ice for 30 minutes
- Heat shock the bacteria in a 37°C water bath for 20 seconds
- Add 950 µL LB to each of the eppendorf tubes
- Incubate for an hour on a shaker in the 37°C room
- Centrifuge at 8000 rpm for 5 minutes
- Decant 900 uL of supernatant and resuspend the rest of the cells
- Take the contents of tubes and spread them on LB Amp plates
- B has a hole, G is barbequed