NucleoSpin C&T

Protocol for the isolation of genomic DNA out of bacteria

Preparation of buffers

Remarks:

Before starting the NucleoSpin C&T Bacteria prepare the following reagents:

If buffer T1 is precipitated, dissolve it by a short incubation at 56C (2-3 minutes)

Prepare buffer B3 in the following manner:

b. Pipet one volume of buffer B2 into four volumes of buffer B1-pipet up and down to mix the buffers completely. (For example: one tissue sample needs 200痞 buffer 3. This means 160痞 B1 and 40痞 buffer B2. Pipet buffer B2 into buffer B1, pipetting up and down to mix the buffers completely.)

The proteinase K must be dissolved in sterile water (1250痞) and should be stored at 4C.

Add 48ml ethanol (96%) to the buffer B5 before use.

Buffers B3 and B5 must be stored at room temperature.

Buffer B1 contains guanidine hydrochloride. Wear gloves and goggles.

Standard Protocol

Attention: Gram positive bacteria is more difficult to lyse. Reagents like lysozyme, lysostaphin, etc. are compatible with this method. For gram positive strains the buffer of step 3 has to be changed (2-4mg enzyme, 20mM Tris/Cl, 2mM EDTA, 1% Triton, pH8). After a 30 minute incubation at 37C follow the protocol with step 4.

1. 1ml of the overnight culture is centrifuged (15 minutes, ~7500 RPM.

2. Make sure that buffers B3, B5 and the proteinase K solution have been prepared according to the remarks above.

3. The pellet of bacterial cells is resuspended in 180痞 buffer T1. Dissolving the pellet is performed by pipetting the solution up and down.

4. Add 25痞 of proteinase K stock solution, mix by vortexing, and incubate at 56C in a shaking waterbath until complete lysis is obtained (~20-30 minutes). Vortex the mixture every 5 minutes briefly. Prepare a 70C waterbath.

5. Add 200痞 buffer B3. Incubate the mixture for 10 minutes at 70C.

6. Add 200痞 ethanol and vortex briefly.

7. Place the NucleoSpin tube into a 2ml centrifuge tube. Apply the sample to the NucleoSpin tube and centrifuge 1 minute at 7500xg (RT). If the sample is not drawn through the matrix completely please repeat the centrifugation step. Discard the filtrate.

8. Add 500痞 buffer B5 (including ethanol) to the spin cup and centrifuge 1 minute at 6000xg (RT). Discard the flowthrough. Repeat this washing step.

9. After the two washing steps with buffer B5, discard the flowthrough, place the NucleoSpin tube again in the centrifuge tube and centrifuge 1 minute at 7500xg (RT) in order to remove buffer B5 completely.

10. Place the NucleoSpin tube into a clean 1.5ml centrifuge tube and elute the DNA with 200痞 preheated (70C) 10mM Tris/HCl, pH=9. Centrifuge for 1 minute at 7500xg (RT). The DNA solution can be used directly for PCR or other enzymatic reactions.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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Updated 1/20/98