MegaLong™ Protocol for Isolation of >100kb Genomic DNA (Cell Culture)
On ice, add up to 2.5x106 cells to a Tube-O-DIALYZER™ and centrifuge at 5,000xg for 5 minutes to pellet cells.
Discard the supernatant.
Add 500µl Nuclei Isolation Buffer.
Invert the tube 2-3 times to suspend the cells, incubate for 10 minutes on ice.
Place the Tube-O-DIALYZER™ cap in a beaker of TE buffer and store at 4°C until required.
Rinse the Tube-O-DIALYZER™ tube with TE buffer.
With a pipette transfer the supernatant to the Tube-O-DIALYZER™, ensuring the settled cellular debris is left behind.
Place a supplied cap on the tube and centrifuge at 16,000xg for 5 minutes to pellet the nuclei.
Carefully discard the supernatant and invert the tube on a paper towel to remove excess supernatant.
Add 70µl Suspension Buffer to the nuclei and gently rock or tap the tube to dislodge the nuclei.
Vortex the LongLife™ Proteinase K and add 10µl to the nuclei.
Add 70µl Digestion Buffer and mix with gentle rocking.
Incubate at 55°C for 2-4 hours with periodic rocking.
Do not vortex.
After digestion is complete, centrifuge the tube for 20 seconds at 1,000g.
Replace the cap with the dialysis cap.
Do not discard the storage cap as this will be required for storage of DNA.
Place the Tube-O-DIALYZER™ upside down in a 50ml centrifuge tube and centrifuge at 1000xg for 30 seconds to bring the sample onto the dialysis membrane.
Remove the Tube-O-DIALYZER™ from the 50ml tube with forceps and keeping it inverted slide into the provided float and dialyze in 500ml 1X TE buffer at room temperature for 18-24 hours with 2-3 buffer changes.
Gently swirl tube to mix contents at each buffer change.
Following dialysis the genomic DNA may be concentrated in the Tube-O-DIALYZER™ using either Tube-O-DIALYZER™ Concentrator (Cat. # 786-144) or Concentrator Solution (Cat. # 786-143).
Simply prepare the Concentrator as per the instructions and invert the Tube-O-DIALYZER™ containing your DNA in the solution.
If concentration is not required or following concentration, centrifuge the tube at 1000xg for 1 minute.
Replace the dialysis cap with the normal cap.
The genomic DNA is now ready for use.
