Mini-prep of plasmids from E. coli
This is a very cheap and rather efficient miniprep though not so clean method to extract plasmids from E. coli
A faster and much cleaner method for performgin minipreps can be performed using the Miniprep kit from Macherey-Nagel.
- Pick some colonies from the LB plate and grow each in a well (96 deep-well plate) with 0.5 ml LB+Ampicillin (75 μg/ml) and incubate overnight at 37°C.
- Transfer 5 μl of each well to a fresh U-bottom plate with 100 μl LB+Amp and place at 37°C.
- With the remaining ~0.5 ml perform a miniprep.
- Centrifuge cells down by spinning plate at 2250x g.
- Pour of the LB and hit the plate upside down on a tissue to remove left over.
- Resuspend cells in 100μl Resuspension buffer (solution #1). Use multichannel when using deepwell plate.
- Add 100μl freshly made Lysis buffer (0.2 N NaOH, 1% SDS; solution #2) and mix.
- Incubate for 5min.
- Add 100μl Neutralisation Buffer (solution #3), mix well and incubate on ice for 5 min.
- Transfer to a 2ml tube, this can be done during the 5 min incubation time.
- Spin down pellet at 12,000 rpm for 5 min.
- Remove the pellet with a toothpick if possible, else, transfer supernatant to fresh tube.
- Add 700 μl 100% ethanol mix and leave at roomtemp for 5 min.
- Spin down DNA at 15,000 rpm for 5min.
- Pour out the supernatant and hit tube on tissue again to remove leftover liquid.
- Rinse the pelleted DNA with 70% -20°C ethanol (in freezer 7).
- Leave the tubes to dry completely
- Add 20μl water to dissolve.
|1M Tris-HCl pH8.0
|0.5M EDTA pH8.0
|5M Potassium Acetate
||~5M Potassium Acetate
|100% Glacial Acetic Acid