ESO wikiadmin at 12:12, 16 June 2025

2025-06-16T12:12:27Z

← Older revision		Revision as of 14:12, 16 June 2025
Line 25:		Line 25:
	* 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 (~~14000rpm~~)		* 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 (~~14,~~000rpm)		* 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

ESO wikiadmin: Created page with "{| class="wikitable" |+ !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 H_..."

2025-05-21T13:11:29Z

Created page with "{| class="wikitable" |+ !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 H..."

New page

{| class="wikitable"
|+
!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 (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/ddH2O for at least 5 min
* Analyse quantity of plasmid DNA preparation

Plasmid-Miniprep (Birnboim – Dooley protocol) - Revision history

ESO wikiadmin at 12:12, 16 June 2025