Genomic DNA isolation experiment based on silica membrane
Genomic DNA isolation experiment based on silica membrane
Silica chemistry has become one of the methods for purifying high-quality genomic DNA from samples such as mouse tail clippings, animal tissues, and tissue culture cells.The Wizard SV Genomic DNA Purification System utilizes a solid-phase silica-filled filter membrane to purify genomic DNA.The Wizard SV Genomic DNA Purification System uses a solid-phase silica-filled filter membrane to purify genomic DNA. This assay is derived from PCR Lab Guide (2nd Edition) by Kang Seed and Lijia Qu.
Operation method
Genomic DNA isolation experiment based on silica membrane
Materials and Instruments
PBS Na2 HP04 KH2PO4 NaCl KC1 Digestive Solution MasterMix Protease K Move I. Materials For more product details, please visit Aladdin Scientific website.
Automatic Positioner P250 Cartridge Tip Flat Seal Vac-Man96 Vacuum Unit Vacuum Pumps
1. buffers, solutions and reagents
1XPBS (for tissue culture cells)
Dissolve the following reagents in 1 L of sterilized deionized water and autoclave. 1XPBS should have a pH of 7.4.
Na2HP04 1.15g
KH2P4 0.2g
NaCl 8g
KC1 0.2g
2. Enzyme and enzyme buffer
(1) Digestion solution MasterMix (for tissues and mouse tails)
Each tissue sample should be mixed with the following components.
Nuclear lysis solution 200ul
EDTA (0.5mol/L) 50ul
Proteinase K(20mg/ml) 20ul
RNAase A solution (4mg/ml) 5ul
(2) Proteinase K (20mg/ml solution; for tissue and mouse tail)
Resuspend Proteinase K in nuclease-free water to a concentration of 20mg/ml, then divide into working volumes based on the average number of samples pretreated at one time. -Store at 20°C and thaw on ice before use, avoiding multiple freezing and thawing.
3. Special equipment
3 units of single-positioned experimental autopositioner (BeckmanCoulter).
4 sets of P250 boxed pipette tips (BeckmanCoulter)
4X4-position laboratory autopositioner (BeckmanCoulter)
96-well Pipette Tips for Paint Wash Automatic Laboratory Positioner (BeckmanCoulter)
Adhesive plate seal (made of metal foil)
Automatic laboratory positioner for heating and cooling (BeckmanCoulter)
Automated machinery for liquid handling (for automated procedures) such as BiomekFx or Biomek2000 automated workstations for experiments (BeckmanCoulter)
Automated experimental positioners for cyclotron oscillation (BeckmanCoulter)
Automatic experimental positioners for pipette tip loading and unloading (BeckmanCoulter)
Vac-Man96 Vacuum Unit (Promega)
Vacuum pump capable of generating 15~20in (lin=2.54cm) Hg column pressure
Vacuum waste collection device (1L size)
Vacuum hose
Water bath, 55°C
4. Other
WiZardSV96 Genomic DNA Purification System (Promega; the system includes a binding wrench, 96-well deep-well plate, DNA elution plate, nuclear lysis solution, 0.5 mol/L EDTA, Wizard SV lysis buffer, WizardSV wash solution, RNAase A solution, and nuclease-free water)
5. Cells and tissues
Samples for genomic DNA purification (mouse tail cuttings, animal tissues or tissue culture cells)
II. Methods
1. Preparation of mouse tail samples and tissue lysates for automated DNA purification
(1) Place a 0.5-1.2 cm long mouse tail excerpt or 20 mg of other tissue in each well of a 96-well deep-well plate. 20 mg of tissue is the maximum allowable sample size, exceeding which may result in the SV96 binding wrench becoming congested during the purification process8 .
(2) After adding 275ul of MasterMix digest per sample, seal the plate with an adhesive seal.
(3) Place the plate in a 55°C water bath for overnight incubation (16~18h).
If purification of genomic DNA is not performed immediately after the overnight digestion with Proteinase K, the sample lysate can be frozen at -70°C for storage.
The frozen lysate must be warmed to 55°C and held for lh before automated purification of genomic DNA.
OPTIONAL: If undigested hair and cartilage remains after overnight digestion with protease, the 96-well plate can be centrifuged at 2000 g to settle the undigested sample, and the supernatant can then be transferred to a new 96-well deep-well plate.
2. Automatic purification of genomic DNA from tissue lysate
After the sample tissue is digested by proteinase K, all the next genomic DNA purification steps can be automated in a liquid handling workbench. After placing the required tools, reagents and warmed tissue lysate plates on the automated liquid handler workbench, the following operations will be performed automatically.
(1) Add 250ul of Wizard SV Lysis Buffer to each sample well.
(2) Pump several times to mix the components in each well.
(3) Transfer the tissue lysate to the wells of the binding plate placed in its empty device by pumping its empty so that all lysate passes over the binding plate to allow genomic DNA to bind to the plate.
(4) Add 1 ml of Wizard SV Wash Solution to the wells of each binding plate.
(5) Pass the wash solution through the binding plate by evacuating it.
(6) Repeat steps (4) and (5) to bring the total number of washes to 3.
(7) After the filter pores are evacuated, continue vacuuming for 6 min to dry the binding matrix.
(8) Prepare its empty device assembler for elution. Remove the binding plate from its empty device, place it into a deep-well elution plate, and then place the binding plate on the elution plate in the same orientation so that the tip of the binding plate is located in the center of the deep-well elution plate.
(9) Add 200 ul of nuclease-free water (room temperature) to each well of the binding plate and incubate for 2 min at room temperature.
(10) Vacuum for lmin to elute purified genomic DNA from the SV96 binding plate into a 96-well deep-well plate.
(11) Repeat steps (9) and (10) to bring the total elution volume to 400ul.
(12) Genomic DMA purification ends with purified samples in 96-well deep-well plates. After sealing the plate with a plate seal, the samples can be stored at -20°C or -70°C.
3. Automatic purification of genomic DNA from tissue culture cells
The following procedure is used to automate the purification of genomic DNA from tissue culture cells cultured in 96-well culture plates.
A maximum of 5X106 cells can be purified; exceeding this number of cells per well may cause the SV96 Binding Plate to become congested during the purification process. The number of cells can be adjusted according to cell type and function.
(1) Cells are washed once with sterilized 1XPBS prior to automated purification and all subsequent steps can be performed in a single well.
All subsequent steps can be automated in a liquid handling workbench. After placing the required tools, reagents and cells (media-free or PBS) on the Automated Liquid Handling Workbench, the following operations are performed automatically.
(2) Add 150ul of WizardSV Lysis Buffer to the washed cells and aspirate.
If the genomic DNA is not to be purified immediately, this step can be stopped after the addition of WizardSV Lysis Buffer, and the sample can be stored at -70°C. After the sample lysate has been melted, the genomic DNA purification can be continued as in step (3).
(3) Transfer the cell lysate to the binding plate in the empty device and pass the wash solution through the plate by evacuating the vacuum, thus
binding genomic DNA to the binding substrate.
(4) Add lml of wash solution to each well of the binding plate and vacuum the wash solution through the plate. 
(5) Repeat step (4) twice so that the number of washes reaches 3.
(6) After the wells are evacuated, continue vacuuming for 6 min to dry the binding matrix.
(7) Prepare vacuum device for elution. Remove the binding plate, place a deep-well elution plate in the vacuum unit, and then place the binding plate on top of the elution plate in the same orientation so that the tip of the binding plate is in the center of the deep-well elution plate.
(8) Add 200ul of nuclease-free water (room temperature) to each well of the binding plate and incubate for 2min at room temperature.
(9) Vacuum for lmin to allow purified genomic DNA to elute from the SV96 binding plate into a 96-well deep-well plate.
(10) Genomic DNA purification is finished, purified samples in 96-well deep-well plates. Seal the plate with a plate sealing strip, and the samples can be stored at -20°C or -70°C.
RNA may be purified along with genomic DNA. To remove the purified RNA, 2ul of RNAase A solution should be added to 250ul of nuclease-free water before eluting the genomic DNA from the column. After elution, the purified genomic DNA should be incubated at room temperature for l0 min. alternatively, RNAase A solution (2ul) can be added after elution.
4. Characterization and quantification of isolated DNA
The yield and quality of WizardSV purified samples can be determined by analyzing A260 and A280 with a spectrophotometer. Depending on the source of the sample (Table 9-2), high molecular weight genomic DNA typically has a yield of 5 to 20 ul,and an A260/A280 value of 1.7 ⫽ 0.08.Figure 9-4 gives an example of a single small processed with the Biomek2000 Automated Mechanical Workbench.
mouse tail samples processed with the Biomek2000 automated mechanical workbench are given in Figure 9-4. The template utility of purified genomic DNA for PCR amplification was also evaluated. PCR amplification of 1ul of purified genomic DNA from mouse tail and tissue culture samples was assessed by amplification of interleukin-1(3(IL-lβ)-specific primers for mouse tail samples, and by amplification of FactorVDNA-specific primers for genomic DNA isolated from HeLa cells (Figure 9-5). 
