96-well E.coli transformation
Material
- DNA sample
- Competent cells
- SOC medium
- Book PCR block
| Check | Reagent | Stock concentration | Final concentration | Volume per 1 reaction (µL) | Volume per _ _ _ _ _ _ _ reaction (µL) | |
|---|---|---|---|---|---|---|
| NEB 5-alpha competent cells | - | - | 10 | |||
| SOC medium | - | - | 100 |
Protocol
- Prepare competent cells on ice to thaw. If you take from multiple tubes, mix in one tue, and aliquot.
- Prepare the reaction mix and competent cells on ice.
- For each sample, add 10µl of homebrew competent NEB5-α cells.
- Incubate on ice for 20mins
- Heatshock on PCR machine @42C for 30secs
- Incubate on ice for 2mins
- Outgrowth by adding 100ul S.O.C. and incubate @37C for 1 hour (static)
- Spot 5 µL of each sample to LB+Amp plates
- Culture/select the transformats by incubating the plate, @37C, O/N.