Small Scale Electrocompetent Prep-Generic: Difference between revisions
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(New page: =Small Scale Electrocompetent Prep - Generic== # Grow up strain to saturation # Innoculate 2YT with 1:30 of o/n. # Grow uninterrupted for 2.5 hours @ 37oC. ##Josh: use 4hrs @ 30C for uberc...) |
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# Immediately add to chilled electroporation cuvette. | # Immediately add to chilled electroporation cuvette. | ||
# Immediately shock | # Immediately shock | ||
# Immediately add 200 uL 2YT, pipette up and down to recover cells, | # Immediately add 200 uL 2YT, pipette up and down to recover cells, move to tube and keep warm | ||
move to tube and keep warm | |||
# Put in shaking 37oC incubator for 1 hr | # Put in shaking 37oC incubator for 1 hr | ||
# Plate everything on appropriate antibiotic plate (allow to dry | # Plate everything on appropriate antibiotic plate (allow to dry | ||
before putting in 37oC incubator) | before putting in 37oC incubator) |
Latest revision as of 17:34, 18 June 2010
Small Scale Electrocompetent Prep - Generic=
- Grow up strain to saturation
- Innoculate 2YT with 1:30 of o/n.
- Grow uninterrupted for 2.5 hours @ 37oC.
- Josh: use 4hrs @ 30C for ubercompetency
- Cool tube/flask; transfer 1.5 mL to chilled epi tube.
- Spin down for 20 sec at 14.1 rpm, discard supe, place immediately on ice.
- Resuspend with .5 mL 10% ice cold glycerol.
- Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
- Resuspend with .5 mL 10% ice cold glycerol.
- Spin down for 30 sec (14.1 rpm), discard supe, place immediately on ice.
- Resuspend in a small volume (90 ul) 10% ice cold glycerol.
- Add ~2uL DNA to cells, pipette & stir to mix.
- Immediately add to chilled electroporation cuvette.
- Immediately shock
- Immediately add 200 uL 2YT, pipette up and down to recover cells, move to tube and keep warm
- Put in shaking 37oC incubator for 1 hr
- Plate everything on appropriate antibiotic plate (allow to dry
before putting in 37oC incubator)