Plasmid-Miniprep (Birnboim – Dooley protocol): Difference between revisions
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Created page with "{| class="wikitable" |+ !Materials |- |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. |- |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>..." |
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* Add 300 µl P2 and invert tube 10x | * Add 300 µl P2 and invert tube 10x | ||
* Add 300 µl P3 solution and invert tube 10x | * Add 300 µl P3 solution and invert tube 10x | ||
* Centrifuge 10 min at 4°C ( | * Centrifuge 10 min at 4°C (12.000rpm) | ||
* Transfer 800 µl supernatant in a new tube and add 0.7 Vol. (560 µl) Isopropanol, invert 3x | * Transfer 800 µl supernatant in a new tube and add 0.7 Vol. (560 µl) Isopropanol, invert 3x | ||
* Centrifuge 15 min at 4°C ( | * Centrifuge 15 min at 4°C (12.000rpm) | ||
* Remove carefully supernatant and wash pellet with 0.5 ml 70% Ethanol | * Remove carefully supernatant and wash pellet with 0.5 ml 70% Ethanol | ||
* Centrifuge 5 min (14,000rpm), remove supernatant | * Centrifuge 5 min (14,000rpm), remove supernatant | ||
Latest revision as of 13:12, 16 June 2025
| Materials |
|---|
| Buffer P1: Solve 6.055 g Tris Base and 3.722g EDTA x 2H2O in 800 ml H20.
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 H20. Add 50 ml 20% SDS. Store at RT. |
| Puffer P3: 294.45g KAc in 500 ml H2O. Adjust pH to 5.5 with glacial acetic acid (about 110 ml acid needed), fill up to 1L with H2O. Store at 4°C. |
| TE-Puffer 10 mM Tris/HCl pH 8.0; 1mM EDTA |
| Isopropanol |
| sterile dd H2O 70% Ethanol |
| For each clone Klon 2 x 1,5ml Eppendorf cups |
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 (12.000rpm)
- Transfer 800 µl supernatant in a new tube and add 0.7 Vol. (560 µl) Isopropanol, invert 3x
- Centrifuge 15 min at 4°C (12.000rpm)
- 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/ddH2O for at least 5 min
- Analyse quantity of plasmid DNA preparation