I am working with Sera-Mag paramagentic beads to isolate whole genomic DNA from human fibroblasts.
I follow the general protocol: bind DNA to beads with PEG/NaCl solution, place on magnetic rack, wash with 70% ethanol, dry, elute with 10mM Tris-HCl (T10).
However, I am having a great deal of trouble eluting. The beads stick to the DNA so tightly that I cannot dissociate the beads to release the gDNA. It looks like a pellet floating inside the T10. I flick the tube as much as possible, but when I put the tube back on the rack to collect the elute, I can see that the beads are still puffy with gDNA (meaning the gDNA is not in solution).
I have tried incubating at 37C for various lengths of time (minutes to days). I have also tried doing a phenol/chloroform extraction first in order to get rid of protein (from cell lysis) that can interfere with the beads. Yet I still encounter this elution issue.
My thought is that the whole gDNA is so long that it is wrapping around the beads and entangling them. However, I need to keep the DNA whole. I do not want to fragment it before using Sera-Mag beads.
Any suggestions would be appreciated.
If you are already performing a Phenol Chloroform extraction do you absolutely need to use the beads to isolate the DNA? You could perform a second extraction to be sure and/or treat the extracted DNA with RNase then precipitate the DNA with a standard EtOH/IPA method. Of course using this method ensure you only dry the pellet long enough for the alcohol to evaporated so you don't encounter issues resolubilising the DNA.
First of all crush your sample, then transfer to 15ml falcon tube, then add 1.5% saline and centrifuge 6000 rpm for 20 mins. After centrifuge, discard the aquas layer, In remain 1 ml pellet, you just add 3ml NLB (Nuclea Lysis Buffer) and 1ml 6M NaCl, and just tab, then centrifuge 4000 rpm for 15 mins. then transfer the Aquas layer to anoother fresh Falcon tubenalong with add equal volume of cold ethanol. Finally, you can get large quantity of DNA...
Thank you for your input, Marcus Hanley and Kamaraj Raju.
Ideally I would not perform any phenol/chloroform extraction and would isolate gDNA directly out of lysis with Sera-Mag beads because 1) Sera-Mag bead extraction is faster and 2) I would like to use its size selection capacity to keep gDNA but get rid of smaller RNAs.
I would like to avoid using RNase A or T1 since my goal is to look at R-loops (RNA:DNA hybrids) in the genome. RNase A can chew away at R-loops in certain conditions and with time.
I would really appreciate input from anyone who has worked with gDNA and Sera-Mag beads before.
Thank you.
Hi Jamie, The method you used works well for purifying DNA from "clean" solutions that do not contain much protein, such as PCR reactions. But you are trying to extract DNA from cells. The puffy beads are most likely an indication of protein binding to your beads which crowd out the DNA. As a result, you won't recover your gDNA. There are several commercial magnetic bead kits out there for DNA purification from cells; the kits formulated for gDNA from blood samples should also work. Typically, they include a cell lysis and protein digestion step with proteinase K and also bind and wash buffers with chaotropes which prevent binding of proteins to the beads. Hope this helps.
@Martina Werner
Thank you for your response.
I lyse my cells with SDS and Proteinase K overnight and then do a phenol/chloroform extraction before putting the DNA on Sera-Mag beads. So, I don't think it is a protein problem. Additionally, I am getting full DNA binding to the beads. I simply cannot elute the DNA into clean water/T10 at the very last step. I attributed the puffiness to the vast amounts of DNA loaded on the beads.
In this case have you considered using more beads and higher elution volumes? you could also heat up the elution buffer to 60 or 70C.
@ Martina Werner
That's a good suggestion. I will try heating to 65C during elution. Previously, I was warming to just 37C.
I went to the manual for the Dynabead blood kit that you mentioned in your previous response and it also says to elute at high temperature (65C for 5min).
So, I was able to elute whole gDNA off of Sera Mag beads by doing multiple serial elutions at 65C for 5 minutes each. It is not fast, but I can make it work.
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