I have a task before me and I would like to know if any of you have walked this path before. I have to isolate genomic DNA from an AIG assay of cancer cells frozen in soft agar at -80C. I need enough DNA to run a PCR. This is what I have done so far:
1. add NaOH/EDTA to get a 50mM/0.2mM solution
2. heat at ~60C for 1 hour (95-10C causes agar to stay in solution and leaves residue in final pellet)
3. add equal volume of 40mM tris at pH5.5
4. Spin at 3500rcf for 5 min, pour off sup into a supercent. tube
5. add slightly more isopropanol than sup volume, spine at 11500rcf for 30 mins.
6. pour off sup, resuspend pellet in 5-700 ul etOH
7. spin for 5 mins at 11,500 rcf
8. aspirate etOH, resuspend in ~10 ul ddH2O
This method has once gained me ~100ng/ul of DNA, but I would like to know if anyone has a more effective method.
Thanks in advance for any help you may provide!