Recovery of Concentrated DNA Fragments from Pulsed Field Gels
Recovery of Concentrated DNA Fragments from Pulsed Field Gels
DNA from low melting point agarose sections is first concentrated by electrophoresis into high percentage agarose gels and then separated by treatment with agarase. The final DNA preparation is purified by microdialysis. This method is very effective in injecting isolated DNA into fertilized mouse eggs (Schedl et al., 1993) or transfected murine embryonic stem cells (Choi et al., 1993). This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Recovery of concentrated DNA fragments from pulsed-field gels
Principle
DNA from low melting point agarose sections is first concentrated by electrophoresis into high percentage agarose gels and then separated by treatment with agarase. The final DNA preparation is purified by microdialysis. This method is very effective in injecting isolated DNA into fertilized mouse eggs (Schedl et al. 1993) or transfected murine embryonic stem cells (Choi et al. 1993).
Materials and Instruments
Agarase Genomic DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Equilibration buffer Injection Transfection buffer
NuSieve GTG Agarose minigel Spot dialysis of membranes Water baths
1. Buffers and solutions
Equilibration buffer (1X TBE, 100 mmol/L NaCl, 30 μmol/L spermidine, 70 μmol/L spermine)
Ethidium bromide (1 μg/ml) or appropriate dilution of SYBR Gold
Injection/transfection buffer (10 mmol/L Tris-Cl (pH 7.5), 0.1 mmol/L EDTA, 100 mmol/L NaCl, 30 mmol/L spermidine, 70 μmol/L spermine)
2. Enzyme and buffer
Agarase
3. gels
NuSieve GTG Agarose minigel (5%)
4. nucleic acids and oligonucleotides
DNA Molecular Quality Standards
Genomic DNA embedded in low melting point agarose plugs
5. Specialized equipment
Membrane for spot dialysis (pore size 0.05 μm)
Water bath set at 65~68℃ and appropriate temperature for agarose enzyme digestion
Methods
1. Prepare low melting point agarose PFGE, which will provide the best resolution for separation of target DNA fragments.
2. Add a gel plug of DNA molecular quality standard and a single digested genomic DNA to each side of the sample filling well of the preparation tank. Add the preparation samples to the preparation tank.
3. At the end of electrophoresis, cut off the lane containing the molecular quality standard and the individual genomic DNA bolus and stain with ethidium bromide or SYBR Gold for 30 min at room temperature, or decolorize in water for 30 min if desired. do not stain the preparation lane.
4. Check the stained sections by UV irradiation and mark the side of the sections with the target DNA.
5. Under normal illumination, reassemble the stained molecular mass standard and individual bolus gels to approximately localize the target DNA in the unstained preparative lanes. Carefully cut off this area with a clean razor and transfer the gel sections to capped polypropylene tubes.
6. Equilibrate the gel sections containing the isolated DNA molecules with 40 ml of equilibration buffer for 20-30 min at room temperature, always shaking the mixture gently.
7. Pour off the buffer and melt the gel at 65-68°C. Rotate the tube while melting to make sure the gel is not too hot. Rotate the tube while melting to ensure complete melting. Record the volume of melted gel sections.
8. with the 5% NuSieve GTG microgel still on the tape-encircled mold, cut away enough volume from the top layer of the gel to accommodate the melted DNA-containing gel slices.
9. Pour the melted gel slice into it and allow it to solidify. electrophoresis the low melting point gel per millimeter for 12 minutes at a constant voltage of 60 V. Concentrate the isolated DNA by swimming it into a 5% gel.
10. After electrophoresis, a thin slice is cut from the center of the gel and stained with ethidium bromide. Determine the length of DNA migration into the gel (usually about 2 mm).
11. Remove the low melting point portion of the gel and cut the 5% gel containing the concentrated DNA as small as possible.
12. Equilibrate the gel sections in 12 ml of 1x Agarase digestion buffer containing 100 mmol/L NaCl, 30 μmol/L spermidine, and 70 μlmol/L spermine. Incubate for 20 min at room temperature with gentle shaking.
13. Pour out the buffer and transfer the gel slices into a microcentrifuge tube and melt at 65~68°C. Transfer the melted gel into a microcentrifuge tube. Transfer the melted gel to a water bath set at a temperature suitable for agarase action (recommended by the vendor).
14. Warm the melted gel for 15 min, and add the appropriate amount of agarase digestion to 5% of the initial gel volume.
15. After digestion, centrifuge the gel in a bench-top centrifuge at maximum speed for 5 min to separate the debris and transfer the supernatant to a new centrifuge tube.
16. Dialyze the resulting supernatant through a 0.05 μm pore size droplet dialysis membrane.
(1) Spot the supernatant in the center of the membrane and float the light side up in 100 ml of injection/transfection buffer.
(2) Dialyze for 1 h at room temperature, change the original buffer, refill with 100 ml of injection/transfection buffer and dialyze for another 1 h. The supernatant should be dialyzed in the center of the membrane.
(3) Transfer DNA to a new microcentrifuge tube.