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SILANE Genomic DNA

互联网2013-11-13

913

实验原理

The Dynabeads SILANE genomic DNA kit provides an excellent tool for isolation of genomic DNA from human blood, following a simple separation protocol. A buffer is first added to the sample for lysis, followed by incubation with Dynabeads MyOne™ SILANE. The beads with bound DNA are easily pulled to the side of the test tube by using a magnet (e.g. Dynal MPC™) and unbound material is removed by aspiration. The magnetic separation also facilitates simple washing and elution of the isolated DNA. Dynabeads magnetic separation technology is easily adapted to automated liquid handling platforms. Please contact Invitrogen Dynal for further information.

实验试剂

Magnetic device (See www.invitrogen.com/magnets for magnet recommendations)

Mixing/rotation device

Test tubes and pipettes

Proteinase K at a concentration of 20 mg/ml

Isopropanol (≥ 99.5%)

Ethanol (96-100%)

实验步骤

1. Preparing the buffers

1) Washing Buffer 1 (genomic DNA) and Washing Buffer 2 (genomic DNA) are supplied as concentrates. To obtain working solutions, isopropanol/ethanol must be added to the two buffers as indicated on the respective labels.

2) Prior to first time use, add 40 ml isopropanol to each of the two supplied bottles of Washing Buffer 1 (genomic DNA) to obtain a working solution.

3) Prior to first time use, add 70 ml ethanol to each of the two supplied bottles of Washing Buffer 2 (genomic DNA) to obtain a working solution.

4) Proceed with isolation of genomic DNA as described below.

2. Isolation of genomic DNA

1) Add 50 μl Proteinase K (20 mg/ml) to a 350 μl blood sample. Vortex 30 seconds (all vortex steps are at maximum speed.) Incubate 2 minutes at room temperature (RT).

2) Add 350 μl Lysis/Binding Buffer (genomic DNA) to the sample and vortex 15 seconds to mix. Incubate for 5 minutes at 55°C and then vortex for 15 seconds. Incubate again for further 5 minutes at 55°C followed by a final vortex for 15 seconds.

3) Add 50 μl resuspended Dynabeads MyOne™ SILANE suspension and vortex for 5 seconds to mix. Add 400 μl isopropanol (100%). Vortex 30 seconds and incubate on a roller for 3 minutes at RT followed by a final vortex for 30 seconds.

4) Place the tube on the magnet and let the Dynabeads collect at the magnet for 2 minutes. Remove the supernatant by using a pipette. The Dynabeads/gDNA will form a nice pellet at the side of the tube, avoid touching this pellet with the pipette.

5) Remove the magnet and add 950 μl Washing Buffer 1 (genomic DNA). Vortex 30 seconds at RT. The bead pellet should easily break into barely visible aggregates.

6) Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute. Remove the supernatant.

7) Repeat steps 5 and 6.

8) Add 950 μl Washing Buffer 2 (genomic DNA). Vortex 30 seconds at RT to resuspend the Dynabeads, and transfer the resuspended bead-solution to a clean tube.

9) Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute (or until the supernatant is clear.) Remove the supernatant.

10) Add 950 ul Washing Buffer 2 (genomic DNA) and vortex 30 seconds.

11) Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute. Remove the supernatant. (Make sure that all the supernatant is completely removed in this last washing step. Use a small pipette tip and make sure that no droplets are left on the tube wall.) Leave the tube on the magnet and let the bead pellet dry for 5 minutes at RT.

12) Remove the magnet and add 100 μl Elution Buffer (genomic DNA). Completely resuspend the Dynabeads by vortexing and/or pipetting for 2 minutes at RT.

13) Place the tube on the magnet and let the Dynabeads collect at the magnet for 1 minute.

14) Transfer the supernatant containing the purified genomic DNA to a clean tube.

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相关问答

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在做DNA印记之前需要哪些试剂来处理genomic DNA?

4 回答 348 围观

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1 回答 3611 围观

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