Genomic DNA Isolation from Soft Agar Colonies - (Mar/13/2014 )
Hello,
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!
Well you can solublise the agarose with Guanidine-HCl (Gu-HCl) or Guanidine Thioisocyanate (GuSCN) at 57 C and use a silicon column to bind the DNA. Once bound, wash the column with an ethanol solution. Then elute the DNA off the column with water or TE.
As an example, you could use the Qiagen DNA purification kit (200bp to ~12kb from my experience). Use QG buffer and heating to 55 C to solublise and lyse the agarose cancer cell plug. I would use 5x volume of QG buffer to plug volume. Then run solution through column, using a centrifuge. You can obtained better binding by passing the DNA QG solution twice through the column. 30 sec centrifuge each. Then wash once with 750ul of fresh QG buffer. Followed by 1 wash of 750ul PE buffer. Then 2 minutes centrifuge to dry the column and remove any PE solution residue. Then add 40ul of warm water or TE, incubate for 5min in 50C incubator and then centrifuge to elute the DNA from column to new tube.
You can also do something similar with the Qiagen miniprep kits. (PB solution instead of QG). From my experience Qiagen miniprep column can purify fragments as large as 70kb. Large fragments need warm elution buffer to get the DNA off the silicon column.
This will give good enough DNA for PCR.
This is a great idea! Thank you! I am wondering, if I use the Qia quick column, will I be able to spin it multiple times to catch all the DNA? I have ~4mL agar/cell suspension, and once I bring it up to 5x in QG, I will have ~20 ml. Do to use multiple columns? If so, how much can I run through before switching columns?
PLGambon on Mon Mar 17 17:10:09 2014 said:
This is a great idea! Thank you! I am wondering, if I use the Qia quick column, will I be able to spin it multiple times to catch all the DNA? I have ~4mL agar/cell suspension, and once I bring it up to 5x in QG, I will have ~20 ml. Do to use multiple columns? If so, how much can I run through before switching columns?
Yes, you can pass the QG + DNA solution through the column multiple times to bind more DNA. However the binding capacity for each qiagen column is said to be about 10ug of nucleotides. So you should spread your QG + DNA solution over several columns. As for eluting the the column, you can run elution buffer (water or Tris) ie 2 times get more DNA off the column and use a spinvac to concentrate the DNA solution.
But, you have a very big plug. How dense are the cells in that plug? You can try calculating an estimate of how much DNA you have. And then match it with the number of column.
I would suggest that you give this method a trial run first to see if you are satisfied with the results.