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How to make competent E.coli cells

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Published on Sep 22, 2013

Protocol for competent E.Coli cells
Needed materials
LB agar plates -- described in my video of transformations here: http://youtu.be/1sjs6wLugDs
Supplemented LB -- prep 500mL of LB as described in my video http://youtu.be/1sjs6wLugDs add MgCl and KCl to a final concentration of 20mM MgCl and 2.5nM KCl, autoclave and store until ready for use.
Buffer TF1 -- 300mL
10mM MES ( 2-(N-morpholino)ethanesulfonic acid) (After adding MES, pH buffer to 5.9)
10mM RbCl
50mM MnCl
10mM CaCl2
pH to 5.8
Filter sterilize, place at +4°C until chilled (usually overnight)
Buffer TF2 -- 100mL
10mM PIPES (1,4-piperazinediethanesulfonic acid)
10mM RbCl
50mM CaCl2
15% glycerol
pH to 6.5
Filter sterilize, place at +4°C until chilled (usually overnight)

Protocol
Day 1:
1.Thaw aliquot of cells on ice, spread on plate, place in incubator at 37°C overnight.
Day 2:
2. Prep supplemented LB, buffers TF1 and TF2.
3. Pick a colony from the plate, inoculate 10mL culture of modified LB, place at 37°C with shaking overnight.
Day 3:
4. Add 10mL starter culture to 500mL of modified LB.
5. Grow culture 3-5 hours at 37°C with shaking monitoring the OD550.
6. When OD550 is close to reaching 0.5 but still less, remove the culture from the shaker, chill on ice for 10min, spin down @ 3000rpm for 5min.
7. Gently resuspend culture in 300mL buffer TF1, chill on ice, spin down @ 3000rpm for 5min.
8. VERY GENTLY resuspend cells in 10mL of buffer TF2, chill on ice for minimum of 15min.
9. Aliquot cells, flash freeze aliquots with liquid nitrogen, store at -80°C
10. Do a test transformation with newly made competent cells (as described in my video http://youtu.be/1sjs6wLugDs ) to assess their compentency
Congratulations you have made competent cells. Stored at -80°C they will remain competent for ~6months

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