Transformation of plasmids in E. coli: Difference between revisions
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Created page with " == Procedure == * Before transformation, appropriate aliquots of competent bacteria should be thawn on ice. * Add the plasmid DNA to 100 μl competent E. coli in a snap-cap tube and mix well. * Incubate the samples 30 min on ice. * Transfer samples for 75 seconds to a 42°C water bath or heating block and quickly back on ice. * Add 1 ml LB-Medium and incubate the bacteria for 1h at 37°C (220 rpm). * Transfer the bacterial suspension from the snap-cap tube to Eppendorf..." |
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{{Protokoll-Mix|Inhalt=Competent E. coli (e.g. strain Nova Blue or BL-21)<br>LB-medium: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, pH 7.0, fill up to 1L | |||
ddH2O<br>LB-plates: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, 10 ml MgCl 2 (1M), 1 g Agar-Agar, pH 7.0, fill up to 1L ddH 2O.<br>Antibiotic: X μg/ml (depending on the resistance gene carried on the plasmid)}} | |||
== Procedure == | == Procedure == | ||
Latest revision as of 08:42, 2 June 2025
Materials
Competent E. coli (e.g. strain Nova Blue or BL-21)
LB-medium: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, pH 7.0, fill up to 1L ddH2O
LB-plates: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, 10 ml MgCl 2 (1M), 1 g Agar-Agar, pH 7.0, fill up to 1L ddH 2O.
Antibiotic: X μg/ml (depending on the resistance gene carried on the plasmid)
LB-medium: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, pH 7.0, fill up to 1L ddH2O
LB-plates: 10 g Bacto-Trypton, 5 g yeast extract, 5 g NaCl, 10 ml MgCl 2 (1M), 1 g Agar-Agar, pH 7.0, fill up to 1L ddH 2O.
Antibiotic: X μg/ml (depending on the resistance gene carried on the plasmid)
Procedure
- Before transformation, appropriate aliquots of competent bacteria should be thawn on ice.
- Add the plasmid DNA to 100 μl competent E. coli in a snap-cap tube and mix well.
- Incubate the samples 30 min on ice.
- Transfer samples for 75 seconds to a 42°C water bath or heating block and quickly back on ice.
- Add 1 ml LB-Medium and incubate the bacteria for 1h at 37°C (220 rpm).
- Transfer the bacterial suspension from the snap-cap tube to Eppendorf-cups, centrifuge at 2500 rpm for 2 min, discard ~900 μl, resuspend the bacterial pellet in the remaining 100 μl, and spread the bacterial suspension with a Drigalski-spatula onto LB agar plates containing appropriate antibiotic for selection.
- Incubate the plates with the lid down in the incubator o/n at 37°C
- For E.coli BL-21, there might be only few colonies as transformation efficiency is regularly low.