Mini-prep of plasmids from E. coli
This is a very cheap and rather efficient miniprep method to extract plasmids from E. coli
- Pick some colonies from the LB plate and grow each in a well (96 deep-well plate) with 0.5 ml LB+Amp (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.
Resuspension buffer
Stock solution
|
Final Concentration
|
for 50ml
|
3.3M Glucose |
50mM |
0.76ml
|
1M Tris-HCl pH8.0 |
25 mM |
1.25ml
|
0.5M EDTA pH8.0 |
10mM |
1 ml
|
H2O |
|
47 ml
|
Lysis buffer
Stock solution
|
Final Concentration
|
for 10ml
|
for 5ml
|
10% SDS |
1% SDS |
1.0 ml |
0.5 ml
|
1.0N NaOH |
0.2N NaOH |
2.0 ml |
1.0 ml
|
H2O |
|
7 ml |
3.5 ml
|
Neutralization Buffer
Stock solution
|
Final Concentration
|
for 50ml
|
5M Potassium Acetate |
~5M Potassium Acetate |
44 ml
|
100% Glacial Acetic Acid |
12% GAA |
6 ml
|