Before starting the NucleoSpin C&T prepare the following reagents:
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 200Ál buffer 3. This means 160Ál B1 and 40Ál buffer B2. Pipet buffer B2 into buffer B1, pipetting up and down to mix the buffers completely.)
a. Preheat buffer B2 for 5 minutes at 70C.
b. Pipet buffer B2 into buffer B1, pipetting up and down to mix the buffers completely. A 200Ál blood sample needs 200Ál of buffer B3 consisting of 160Ál of buffer B1 and 40Ál of buffer B2.
c. The RNase (heat treated), must be added to buffer B4 before use. After the addition of the RNase the buffer B4 should be stored at 4C.
d. The proteinase K must be dissolved in sterile water (1250Ál) and should be stored at 4C.
e. Add 48ml ethanol (96%) to the buffer B5 before use.
f. The buffers B3 and B5 must be stored at room temperature.
g. If the volume of the blood sample is less than 200Ál add the appropriate volume of PBS buffer to the sample. For the preparation of PBS dissolve 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g of KH2HPO4 in 800ml of distilled water. Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Dispense the solution into aliquots and sterilize them. Store at room temperature.
1. Prepare a 70C waterbath.
2. Equilibrate the blood sample to room temperature.
3. Make sure that buffers B3, B4 and B5 and the proteinase K solution have been prepared according to the remarks above.
4. Pipet the blood sample into a 1.5ml centrifuge tube. If the volume of the sample is less than 200Ál add the appropriate volume of PBS buffer (see remarks above).
5. Add 25Ál proteinase K solution and 200Ál buffer B3 to the sample and vortex the mixture until any pellet is dissolved.
6. Incubate the mixture at 70C for 10 minutes.
7. Cool the mixture to room temperature, add 100Ál of buffer B4 (including RNase), vortex and incubate the sample 5 minutes at room temperature.
8. Add 210Ál isopropanol (or ethanol) to the sample and vortex again.
9. 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 repeat the centrifugation step. Discard the filtrate.
10. Add 500Ál buffer B5 (including ethanol) to the spin cup and centrifuge 1 minute at 6000xg (RT). Discard the flowthrough. Repeat this washing step.
11. After the two washing steps with buffer B5 discard the flowthrough. Place the NucleoSpin tube into the centrifuge tube again and centrifuge for 1 minute at 6000xg (RT) in order to completely remove buffer B5.
12. Place the NucleoSpin tube in a clean 1.5ml centrifuge tube and elute the DNA with 200Ál preheated (70C) 10mM Tris/HCl, pH 9. Centrifuge for 1 minute at 6000xg (RT).
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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