View source for Plasmid-Miniprep (Birnboim – Dooley protocol)

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!Materials
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|Buffer P1: Solve 6.055 g Tris Base and 3.722g EDTA x 2H<sub>2</sub>O in 800 ml H<sub>2</sub>0. 
Adjust pH to 8.0 with 1M HCl, fill up to 1L. 
Per 1 ml buffer P1, add 100 µg RNase A. Store at 4°C.
|-
|Buffer P2: Solve 8.0 g NaOH in 950 ml H<sub>2</sub>0. Add 50 ml 20% SDS. Store at RT.
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|Puffer P3: 294.45g KAc in 500 ml H<sub>2</sub>O. Adjust pH to 5.5 with glacial acetic acid (about 110 ml acid needed), fill up to 1L with H<sub>2</sub>O. Store at 4°C.
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|TE-Puffer 10 mM Tris/HCl pH 8.0; 1mM EDTA
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|Isopropanol
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|sterile dd H<sub>2</sub>O 70% Ethanol
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|For each clone Klon 2 x 1,5ml Eppendorf cups
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== Procedure ==

* Resuspend completely bacterial pellet in 300µl P1 solution
* Add 300 µl P2 and invert tube 10x
* Add 300 µl P3 solution and invert tube 10x
* Centrifuge 10 min at 4°C (14000rpm)
* Transfer 800 µl supernatant in a new tube and add 0.7 Vol. (560 µl) Isopropanol, invert 3x
* Centrifuge 15 min at 4°C (14,000rpm)
* Remove carefully supernatant and wash pellet with 0.5 ml 70% Ethanol
* Centrifuge 5 min (14,000rpm), remove supernatant
* Centrifuge pellet for additional 2 min at 14,000rpm
* Dry the pellet at 37°C (~ 15 min)
* Solve the pellet in 40 µl TE Puffer/ddH<sub>2</sub>O for at least 5 min
* Analyse quantity of plasmid DNA preparation