实验试剂

Dynal® magnet: see www.invitrogen.com/magnets-selection.

Sterile, round-bottomed 2 ml microcentrifuge tubes.

Micropipettors and sterile, disposable pipette tips.

65°C water-bath or heating block (only needed for elution).

PBS pH 7.4 (Phosphate Buffered Saline - for buffy coat, see below).

Sterile and PCR-grade water for dilution of supplied buffers.

实验步骤

1.        DNA Isolation Protocol from 100 μl Blood

1)        In a 2 ml round-bottomed microcentrifuge tube mix 100 μl anti-coagulated blood with 1 ml pre-diluted Red Cell Lysis Buffer (diluted 1:5 from the original concentration supplied, stored at 2-8°C and used cold). If less than 10 μl of blood is used as starting material, this Red Cell Lysis step can be omitted from the protocol.

2)        Leave at room temperature with occasional shaking of the tube until the red cells have lysed and the solutio  is clear. Red Cell Lysis should give a clear solution of bright red colour (lysis is usually complete in 5 minutes).

3)        Centrifuge for 30 seconds at 13,000 rpm in a microcentrifuge to pellet the white blood cells. A small, white or light grey haemoglobin-free pellet of WBC should be visible. Remove and discard supernatant.

4)        Immediately add 200 μl (1 unit) of Dynabeads to the pellet in a single rapid pipetting action to mix the components. Do not vortex or mix further.

5)        Leave tube undisturbed at room temperature for 5 minutes.

6)        Place tube on the magnet. Allow the DNA/Dynabeads complex to move to the tube wall after 1 minute carefully pipette off and discard supernatant. The DNA/Dynabeads complex will have a brown gelatinous appearance. During this step and subsequent washes, ensure that the complex is not disturbed or broken up.

7)        Remove the tube from the magnetic field and wash the DNA/Dynabeads complex with 1 ml 1x Washing Buffer (brought to room temperature prior to use). The buffer should be added in a single, rapid pipetting action washing theNA Note: complex off the wall of the tube. Do not break up the complex as this will reduce the DNA yield.

8)        Replace the magnetic field, let the DNA/Dynabeads complex move to the side of the tube and after 1 minute, or when supernatant has cleared, pipette off and discard the supernatant.

9)        Repeat step 7-8 twice, each time ensuring that the supernatant is completely removed and the DNA/Dynabeads complex is intact.

10)    Remove the tube from the magnetic field and thoroughly resuspend the DNA/Dynabeads complex in 200 μl Resuspension Buffer or a volume suitable for your PCRapplication.  This suspension can be used directly for PCR-amplification.

11)    If required, elute the DNA by incubation at 65°C for 5 minutes. Immediately place the tube in the magnet for 30 seconds or until the suspension has cleared, pipette off supernatant and transfer to a clean tube. Elution should give a clear, noncoloured, bead-free solution.

12)    1% of the DNA/Dynabeads complex will be sufficient DNA-template for one 30-35 cycle PCR-reaction. Up to a maximum of 5% of the complex or up to 50% of eluted DNA can be used as starting material for PCR (50 μl volume).

13)    If determination of DNA concentration by absorbance measurements at OD260 is required, the DNA will first have to be eluted off the beads. Ensure that there are no beads left in the solution as they will interfere with the spectrophotometrical readings in an unfavourable way. Eluted/non-eluted DNA from 100 μl of blood is sufficient for at least 100 PCR-reactions. The DNA/Dynabeads suspension after step 10 can be used directly for PCR-amplification. To prevent autocatalytic degradation if the DNA is to be stored at 4°C or -20°C elute the DNA a  described in step 11.

2.        DNA Isolation Protocol from 500 μl Blood

1)        Take 0.5 ml anti-coagulated whole blood in a 2 ml round bottomed microcentrifuge tube.

2)        Add 1 ml Red Cell Lysis Buffer B1, mix and invert tube several times until the red cells have lysed and the solution is clear.

3)        Microcentrifuge for 10-30 seconds at 13,000 rpm to pellet WBC. Remove and discard the supernatant.

4)        Vortex tube to resuspend WBC pellet, add 1 ml Red Cell Lysis Buffer B2 and mix. If lumps are observed, leave to settle for one minute and transfer supernatant to a new tube.

5)        Microcentrifuge for 10-30 seconds at 13,000 rpm, remove and discard supernatant. A small, white or light grey, haemoglobin-free pellet of WBC should be visible.

6)        Vortex the tube and add 200 μl (1 unit) of Dynabeads to the WBC pellet in a single rapid pipetting motion. Immediately aspirate the contents of the tube (using the same pipette tip) and transfer to a clean 2 ml tube. No incubation is necessary, but for maximum yield incubate for 5 minutes at room temperature. Do not vortex or mix further. At this point a complex of DNA/Dynabeads with a brown, gelatinous appearance should be visible.

7)        Place the tube in the magnetic field and after 30-60 seconds, carefully aspirate and discard supernatant. During this step and subsequent washes, keep the complex intact.

8)        Remove the magnetic field and add 1 ml 1x Washing Buffer. Use a single, rapid pipetting action to flush the complex from the tube wall. Do not break up the complex.

9)        Replace the magnetic field and after 30 seconds, aspirate and discard the supernatant.

10)    Wash the complex twice by repeating steps 8 and 9 ensuring the supernatant is completely removed and the complex is intact.

11)    Remove the magnetic bar. Add 200 μl Resuspension Buffer and disrupt the complex by repeatedly pipetting up and down. This suspension can be used directly for PCR-amplification

12)    If elution is required, incubate at 65°C for 5 minutes and place the tube directly in the magnetic field. After 1 minute, transfer supernatant with the DNA to a new tube. Elution should give a clear, bead-free solution.

13)    10-20 μl of the isolated and eluted DNA can be used to prepare a working dilution sufficient for 100 x 10 μl PCR reactions. The remainder may be stored frozen.

14)    If determination of DNA concentration by absorbance measurements at OD260 is required, the DNA will first have to be eluted off the beads. Ensure that there are no Dynabeads left in the solution as the beads will interfere with the spectrophotometrical readings.