Cheap miniprep

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.

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