An experimental method for the isolation of genomic DNA based on magnetic beads
An experimental method for the isolation of genomic DNA based on magnetic beads
The Wizard Magne Sil Genomic DNA Purification System uses a solid-phase paramagnetic silica pellet to purify genomic DNA, replacing vacuum filtration and centrifugation-based forms of DNA purification, but also making sample processing more economical and efficient. This experiment is from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
An experimental method for the isolation of genomic DNA based on magnetic beads
Materials and Instruments
Whole blood Oral smear or sample contaminant Dithiothreitol Ethanol Isopropanol Lysis buffer Move Method 1: High-yield isolation of genomic DNA from 20ul of whole blood using a liquid handling automated mechanical workstation For more product details, please visit Aladdin Scientific website.
Automated Positioner P250 Cartridge Pipette Tips Blood Genome High Yield Lines 96-well Collection Plates BiomekFX Automated Mechanical Workstations Separation Units Heat Transfer Plates Reservoirs Micropipette Tips Centrifuge Baskets Heating Blocks Magnetic Separation Racks DNAIQ Systems
This method is used to process 20ul blood samples in 96-well plates mounted on liquid handling automated machinery such as the BiomekFX and Biomek2000 experimental automated workstations (BeckmanCoulter). If a single plate is processed with a single head device, the time required is less than lh.
Mixing blood with descaler-promoter and lysis/binding buffer disrupts both the cells and their nuclear membranes, and the
denaturation of proteins. Released DNA is adsorbed to the surface of MagneSil particles in the lysis/binding buffer, while magnetic beads with the target complex adhered to them are captured by an external magnetic field to the side of the sample plate wells, and the lysate is subsequently removed as a waste solution. Residual contaminants were removed by the following series of washes: first with lysis/binding buffer, then with a salt wash solution, and finally with an ethanol/salt wash solution to remove the last remaining microvarieties of ferrous heme. After drying of the magnetic beads, the DNA was eluted with TE buffer or water on a heat transfer plate.
I. MATERIALS
1. Special equipment
Three single-unit experimental autopositioners (BeckmanCoulter)
4 sets of P250 boxed pipette tips (BeckmanCoulter)
4X4 position laboratory autopositioner (BeckmanCoulter)
96-well collection plate (Promega, A9161 or similar)
96-well pipette tip washing automatic positioner for experiments (BeckmanCoulter)
BiomekFX automated mechanical workstations fitted with a single compartment and a 96-well pipette head (BeckmanCoulter)
Deep-well MagnaBot 96-well magnetic separation device (Promega)
Deep-well multiwell plates (Marsh, AB-0932 [2.2 ml] and AB-0787 [1.2 ml] or equivalent)
Heat transfer plates (Promega, Z3271 or similar).
Positioners for heating and cooling automated experiments (BeckmanCoulter)
MagnaBot side bar, l/8in (lin=2.54cm) (Promega)
Rotary Oscillation Automatic Laboratory Positioner (BeckmanCoulter)
96-well reservoir with tapered bottom (InnovativeMicroplates, S30014 or similar)
Pipette tip loading and unloading automatic laboratory positioner (BeckmanCoulter)
2. Other
MagneSil Blood Genome High Yield Dong System (Promega; the system includes an ethanol-washing solution, anti-foam reagent, cleavage and other reagents).
Lysis Buffer, Salt Wash, Elution Buffer, and MagneSil Paramagnetic particles).
3. Cells and Tissues
Whole blood, oral smears or sample contaminants 
II. Methods
1. 400ul of lysis buffer and 40ul of MagneSil granules were added to 200ul of blood sample to lyses leukocytes using vigorous mechanical mixing and to bind the released DNA to the magnetic particles (PMP).
2. Capture the particles with a MagnaBot magnetic field and discard the liquid.
3. Wash the PMP thoroughly with 360ul of lysis buffer.
4. Capture the pellet with MagnaBot magnetic field and discard the liquid.
5. Wash PMP thoroughly with 360ul salt buffer.
6. Capture the particles with MagnaBot magnetic field and discard the liquid.
7. Repeat steps 5 and 6 for a total of 2 salt washes.
8. Wash the PMP thoroughly with 360ul of ethanol wash buffer.
9. Capture the particles with MagnaBot magnetic field and discard the liquid.
10.Repeat steps 8 and 9 for a total of 3 washes with Ethanol Wash Buffer.
11.Dry the PMP on a heating block to remove all residual ethanol wash buffer.
12. mix vigorously with 210ul of elution buffer on a heating block at 80°C to elute the DNA. the temperature in the wells is about 65°C.
13. store the eluted DNA at -20°C.
Method 2: DNAIQ isolation of genomic DNA from contaminants and oral smears
The DNAIQ isolation system utilizes a novel approach to DNA isolation. The MagneSil chemistry allows for the capture and release of certain amounts of DNA from a variety of samples. The particles have the ability to carry a certain amount of DNA, but if too much DNA is present, only a certain amount of nucleic acid can be bound (Figure 9-6). Therefore, this method is usually used for experiments that isolate approximately l00ng of DNA, regardless of the sample size. The DNA is eluted with 100ul of elution buffer to a final concentration of lng/ul. The resulting DNA isolated by this method does not need to be purified for quantification. Figure 9-7 gives the flow of this scheme. 
I. Materials
1. buffers, solutions, and reagents
Dithiothreitol (DTT), lmol/L
Ethanol, 95%?100%.
Isopropyl alcohol
Lysis buffer
Determine the total amount of lysis buffer to be used (Table 9-3). Add 1ul of 1moL/L DTT to each 100ul of Lysis Buffer (supplied with the DNAIQ System), invert several times to mix, and mark and date the addition of DTT. Lysis buffer should not be stored at room temperature for more than 1 month. 
2. Special equipment
Aerosol-resistant micropipette tips
DNAIQ Centrifuge Basket (Promega, V1221)
Heating Blocks
MagneSphere Technology Magnetic Separation Racks (Promega)
Microcentrifuge tubes, 1.5 ml conical (Promega, V1231)
3. Other
DNAIQ System (Promega; the system includes resin, lysis buffer, 2X wash buffer, and elution buffer)
Microcentrifuge tube, 1.5 ml (Promega, V1231)
II. Methods
1. Place the sample (see Table 9-3) in a 1.5 ml microcentrifuge tube. Remember: the maximum amount of DNA that can be captured with the recommended amount of resin is approximately l00ng.
2. Add the appropriate amount of prepared Lysis Buffer. Different volumes of Lysis Buffer are required for different samples, see column 2 (Lysis Buffer 1) in Table 9-3 to determine the appropriate amount. Cap the tube and place the tube on a heating block at 95°C for 30 min. For small spots, an alternative is to place the contaminated material on a DNAIQ column with a 1.5 ml microcentrifuge tube and add 100?150ul of Lysis Buffer to the column. Carefully cap the tube and heat at 95°C for 30 min# If using the microtubes and columns shown in Figure 9-7, most of the buffer will remain in the centrifuge tubes and proceed to step 4. This method should not be used if the volume of Lysis Buffer required for the sample exceeds 150 ul.
3. Remove the tube from the heating block and transfer the Lysis Buffer and sample to the centrifuge column in a 1.5 ml micro conical centrifuge tube. It is important to centrifuge the Lysis Buffer with the contaminated matrix to obtain the maximum amount of contaminant.
4. Centrifuge at maximum speed for 2 min at room temperature and remove the column.
5. Vortex the resin reservoir bottle at high speed for l0s to mix thoroughly and add 7ul of resin to the DNA solution, keeping the resin in a resuspended state when adding to obtain the same results.
6. Vortex the sample/lysis buffer/resin mixture at high speed for 3s and hold at room temperature for 5 min.
7. Vortex the tube at high speed for 2s and place it on a magnetic rack for immediate separation.
8. Carefully discard all solution without damaging the resin on the side of the tube.
9. Add 100ul of lysis buffer, remove the tube from the rack and vortex at high speed for 2s.
10. Reposition the tube on the rack and discard all of the lysis buffer.
11. Add 100ul of 1X Wash Buffer, remove the tube from the rack and vortex for 2s at high speed.
12. Reposition the tube on the magnetic rack and discard all of the Wash Buffer.
13. Repeat steps 11 and 12 twice for a total of 3 washes, removing all solution after the last wash.
14. Uncap the tubes and air dry the resin on the magnetic rack for 5 minutes.
15. Add l00ul of Elution Buffer.
16. Close the cap and vortex the tube at high speed for 2s, then place the tube at 65°C for 5 min.
17. Remove the tube from the heating block and vortex at high speed for 2 s. Immediately place the tube on a magnetic rack.
18 Transfer the solution to a new container. the concentration of DNA is approximately 1 ng/ul and can be used directly in the amplification reaction. the DNA solution can be stored for a short period of time at 4°C or for a long period of time at -20°C or -70°C. the DNA solution can be stored at 4°C for a short period of time or at -20°C for a long period of time.