Strepts and Qiagen

Fri Sep 15 16:12:04 EST 1995

In article <434v53$q5g at>, rdaines at (R Daines)

> Does anyone have any experinece in isolating plasmids from Streptomyces
> sp. using Qiagen columns?

Preparation of plasmid DNA from Streptomycetes 
miniprep culture (10 ml culture TSB or YEME)

This procedure has been adapted by customers. It has not 
been thoroughly tested and optimized by QIAGEN. Please 
be sure to read the QIAGEN Plasmid handbook and the 
comments to the protocols carefully before starting your 
experiment. Follow QIAGEN E. coli plasmid mini protocol 
with the following modifications:

1. 	Centrifuge the culture.

2.	Wash pellet once with 10.3% sucrose solution.

3.	Centrifuge again.

4.	Keep the culture at minus 20¡C for at least 1 to 2 hours, or 

5.	Add 0.75 ml buffer P1 which additionally contains 3 mg/ml 

6.	Incubate for 15 to 30 minutes at 37¡C
7.	Add 0.75 ml buffer P2.

8. 	Incubate at room temperature for at least 10 minutes.

9.	Add 0.75 ml buffer P3.

10. 	Follow the QIAGEN-tip 20 protocol (QIAGEN Plasmid Mini)
	 at the equilibration step. Apply 2 x 1 ml supernatant to a 
	QIAGEN-tip 20 column.

11. 	After the precipitation with isopropanol wash once with 
	75% EtOH.

12.	Dissolve the pellet in 30µl water. 

Notes:	3 µl are sufficient for one restriction digestion.
		Yield: Approximately 20 µg DNA

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