Difference between revisions of "Cheap miniprep"
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− | == Mini-prep of plasmids | + | == Mini-prep of plasmids using kit (''E. coli'') == |
Using kit from Macherey-Nagel | Using kit from Macherey-Nagel | ||
Revision as of 14:56, 3 March 2020
Mini-prep of plasmids using kit (E. coli)
Using kit from Macherey-Nagel
- Grow cells over night in 5ml liquid LB medium
- Spin down cells from 2 ml in 2ml tube at 14000xg for 1 min
- remove supernatant.
- Repeat with another 2ml in the same tube
- Add 150µl resuspension buffer and re-suspend all cells
- Add 250µl lysis buffer (blue) and invert 5 time
- Add 350µl neutralisation buffer and invert till the whole liquid is no longer blue
- Spin down the cell debris and genomic DNA at 20.000xg for 3 min
- Prepare the columns in tubes or on the Vacuum manifold
- add liquid without any pellet (better to add less supernatant than accidentally add gDNA)
- Spin down (1min at 13000xg) or vacuum and remove flow through
- Add 450µl washing solution and spin down or vacuum
- Remove flow through and spin down to remove last bits of liquid
- Move column to 1.5ml collection tube.
- Add 30µl elution buffer AE
- Incubate for 2min
- Spin down (1min at 13000xg)
- Add 30µl elution buffer AE
- Incubate for 2min
- Spin down (1min at 13000xg)
- Label tube and trash column
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 |