Follow this protocol to extract the DNA molecules of a certain size range from a DNA mixture.

Equipment

• Magnetic rack
• Use the proper rack for your tube format. For example, if you are using 1.5 mL "eppendorf" tube, use the 1.5 mL magnetic rack.
• If you are using 96 wells plate, use the magnetic rack for 96 wells plates.
• Pipette set for 1000, 200 and 20 microliter
• Timer

Consumables

• Magnetic beads for DNA purification
• Ethanol 80%
• Water molecular grade, nuclease-free
• Pipette tips
• 1000 microliter
• 200 microliter
• 20 microliter
• Empty test tubes of your format (for example, 1.5 mL "Eppendorf" tubes, etc.)

Calculations

1. Decide the length range of the DNA molecules that you want to preserve
2. Calculate the beads volume needed for the longer DNA removal, according to the table:

DNA molecule size	Beads to sample volume ratio	Add this volume of beads (microliters) to the 50 μL sample
100	1.5 x	75
200	1.0 x	50
300	0.8 x	40
400	0.7 x	35
600	0.6 x	30
700	0.5 x	25
1300	0.4 x	20

For example, if  you want to preserve DNA of the length 200-600 bp, then you need to remove everything longer than 600 bp. You should therefore use the sample to beads ratio of 0.6x. For the 50 microliters sample solution, you should add 30 microliters of the beads suspension.
1. Calculate the beads volume needed for the shorter DNA removal, according to the table above. Using the example above, you need to remove everything that is shorter than 200 bp. You should use the sample to beads ratio 1.0x, including the beads volume used to remove longer DNA! That is, you should add the total of 50 microliters of beads, but you already added 30 microliters at the previous stage of longer DNA removal. Therefore, at the shorter DNA removal stage, add 50-30 = 20 microliters of the beads suspension.

Procedure

The volumes are given for the size selection of the fragments 200-600 base pairs from the 50 microliters of sample solution.

1. Take the suspension of the magnetic beads for DNA purification from the fridge
2. Vortex thoroughly until the suspension is homogenous
3. Bring the magnetic beads suspension to the room temperature
4. Add the magnetic beads for the longer DNA removal. In our example, add 30 microliters of beads suspension
5. Vortex the sample
6. Incubate on bench for 5-15 minutes, so the DNA absorbs on the beads
7. Place the sample into the magnetic rack
8. Wait for the solution to clear (30 seconds - 2 minutes)
9. Label new tubes and place them in a tube rack
10. Transfer the supernatant to the newly labelled tubes. Using our example, the supernatant contains DNA shorter than 600 bp.
11. Discard the tubes with magnetic beads
12. To the tubes with the supernatant, add the magnetic beads suspension for the shorter DNA removal. In our example, add 50 - 30 = 20 microliters of the magnetic beads suspension
13. Vortex the sample
14. Incubate on bench for 5-15 minutes, so the DNA absorbs on the beads. The short DNA remains in the solution
15. Place the sample into the magnetic rack
16. Wait for the solution to clear (30 seconds - 2 minutes)
17. Pipette the supernatant out while keeping the tubes on the magnetic rack. The supernatant contains the short DNA and the low molecular weight impurities. The desired DNA is absorbed on the beads.
18. Perform two wash steps while keeping the tubes in the magnetic rack:
1. Add 250 microliters of 80% ethanol.
2. Wait for 30 seconds
3. Discard the supernatant
4. Add 250 microliters of 80% ethanol
5. Wait for 30 seconds
6. Discard the supernatant
7. With 20 microliter pipette, remove all the remaining ethanol from the bottom of the tube
19. Leave test tubes with the beads open for 3-5 minutes on the bench to dry out the ethanol. Don't dry the beads completely, as the DNA yield will decline.
20. Elute the DNA from the beads:
1. Remove the tubes from the magnetic rack
2. Add 20-30 microliter of water or buffer of choice to the tubes with the beads
3. Vortex the tubes until all the beads are in suspension
4. Wait for 5-15 minutes on the bench to complete the elution. The desired DNA is now in the solution
21. Place the test tubes into the magnetic rack
22. Wait for the solution to clear (30 seconds)
23. Transfer the supernatant (that contains DNA) to the fresh tubes
24. Discard the tubes with the beads
25. Store the rest of the magnetic beads suspension in the +4 degrees C fridge.