NucleoSpin C&T

Protocol for the isolation of genomic DNA from tissue

Preparation of buffers

Remarks:

Before starting the NucleoSpin C&T Tissue 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

1. Tissue: Cut 25mg of tissue into small pieces, place in 1.5ml centrifuge tube and and 180痞 buffer T1.

Liquid nitrogen samples: Transfer the powder from 1cm of tissue to a 1.5ml centrifuge tube and add 180痞 buffer T1.

Mechanical homogenized samples (Polytron, Ultra Turrax): Add 25mg of tissue to a 1.5ml centrifuge tube and add 180痞 PBS buffer. After homogenization proceed with step 3.

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

3. Add 25痞 of proteinase K stock solution, mix by vortexing, and incubate at 56C in a shaking waterbath until complete lysis is obtained (~1-3h). Additional 3-4 times vortexing every 10-15 minutes leads to shorter lysis times. At the end of this incubation prepare a 70C waterbath.

4. Add 200痞 buffer B3 to the sample, vortex the mixture and incubate it at 70C for 10 minutes.

5. Add 210痞 ethanol to the sample and vortex immediately. If a white precipitate is obtained after the addition of ethanol, make sure it is all applied to the NucleoSpin cup.

6. Place the NucleoSpin tube into a 2ml centrifuge tube. Apply the sample to the NucleoSpin tube and centrifuge 1 minute at 6000xg (RT). If the sample is not drawn through the matrix completely please repeat the centrifugation step. Discard the filtrate.

7. 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.

8. 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 6000xg (RT) in order to remove buffer B5 completely.

9. 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 6000xg (RT). A five minute incubation at 60-70C of the NucleoSpin column and the buffer before centrifugation leads to a better yield.

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

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Updated 1/20/98