Ammonium acetate precipitation of NaOH treated DNA - (Sep/01/2005 )
i have some problems with the last step of my bisulfite sequencing.
There you have to desulphonate the treated and again purified DNA by 0,3M NaOH and then to precipitate by 2-3M ammonium acetate and 2,5v Ethanol. My problem is that this precipitation step doesnt work at all. I hav 500ng DNA and add 10µg of tRNA and everything is cooled. After all the tRNA is precipitated but the DNA is gone. ???
I did Tests with DNA which was not treated with NaOH and there the precipitation works well.
Does the ammonium acetate has to have a certain pH?
Has anybody an idea?
Make sure 2-3 M ammonium acetate is the final concentration.
What method do you use for the purification before desulphonation? DNA may be lost during the purification step.
how are you determining your DNA is gone?
normally you don't see a DNA pellet after conventional modification. it's there but in very little quantity!
After the bisulfite treatment I purify by a normal silica based system (NucleoSpin from Macherey Nagel).
Since I use 500ng of genomic DNA its quite good visible on agarose gel. I checked before bisulfite modification, after purification and after precipitation, but there its gone. I used 2-3M final concentration of ammoniumacetate. Acually I know that even after the final desulphonation step the DNA is still there because I can purify it by Microcon (these are expensive filter devices from Amicon).
I have the strong feeling that something is wrong with the pH.
From your answers I conclude that you don't adjust the pH of your ammonimum acetate!?
I did tests with untreated genomic DNA. I added NaOH to 0,3M final conc. heated it for 15min at 37°C, cooled it down again and tried to precipitate it. It did not work at all. Of course I did a control with DNA which I did not treat with NaOH and there it works well!
Do I overlook sth.?
I do pH my ammonium acetate,
after bisulfite treatment, you will not see the DNA on the gel, as bisulfite degrades DNA is such a way that you need to perform PCR to obtain the region of interest, two rounds of PCR is ideal though.
it has been reported in the literature that bisulfite treatment will degrade approximately 80-90% of your starting DNA. Because of this you are not seeing your DNA, but trust me it should be there, even if you can't see it on a gel.
Kits claim to be able to perform modification without the loss of DNA, I have tested such a kit called methyleasy and its great, if only i know what they use to ensure no degradation. but i guess that is why it is patented and you have to buy from them.
To what pH? pH5?
After bisulfite treatment (16h, 55°C) and purification but before desulphonation i still see maybe half of it. i use the sodium bisulfite from aldrich (243973) which replaced the one from sigma (S-8890). Did anybody ever tried this one? Maybe something is wrong with it? Do you actually store it specially?
What conc. of hydroquinone do you use? In my protocol from Liu et al. they use only 1,8mM.
hi here is a doc I have attached to a previous post that I can not find now!!
I use the same bisulifte powder from Sigma as you, we store it at room temp on the lab bench.
We use 100mM hydroquinone a slight modification to the published protocols.