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 .
