Let us assume that I lyse some cell and now all the components are free.
Now if I wish to just pull down all the DNA and RNA (nucleic acids) what type of chemistry can work? (Considering the fact that we can use any other proteins or compounds which can bind to nucleic acids and thus pull them via the bound protein or compound)
Give your valuable molecular suggestions.
Hello Alim.
I here by meant that if by any mild mechanism i manage to break all the cellular membranes of a cell and thus i have a lysate with all biomolecules. now from that lysate i just wish to PULL DOWN all the nucleic acids.
When I say pull down I mean to get information on any compound/chemical structure/protein which can bind to DNA in the lysate and then we pull the DNA by using the COMPOUND/CHEMICAL STRUCTURE/PROTEIN as the source. Its similar to a Biotin-Streptavidin/ Histidine- Nickel chemistry. But in my query it should pull down DNA.
And I dnt wanna do any sequential extractions here.
Step 1: Lysis
Step 2: Add SOME THING which will bind to DNA
Step 3: Pull down the DNA using the SOME THING.
Thats it so I want to know what is that SOME THING and what is the source to pull down.
I know its a very basic question but is there a biological solution for it.
Yes Alim
Its more of Chemistry I am also looking for. I will be using the DNA for PCR/Real Time PCR.
Ok let us assume that in the buffer I will lyse my cells I include proteases which will free proteins bound to DNA . Now that DNA is partially free from proteins. I have DNA alone right.
and I might be wrong but ChIP is more for identifying DNA binding proteins but not to pull down DNA.
Lets see if we get any chemistry inputs here to further discuss.
Yes. I had few ideas but I guess they wont work. to share they are like
As Etbr / Sybr has DNA binding ability. any possibility of chemically modifying ETBR/SYBR to conjugate with a nanoparticle/magnetic component thus pull the DNA out using ETBR/SYBR.
But Why i feel they wont work is even if modify ETBR/SYBR so as to make it bind to DNA. how will i get rid of ETBR/SYBR after i pull down and if i find a way to get rid of ETBR/SYBR from DNA after pulling down then my idea might work
I just found this journal whose full text i am not able to get but the title says
Removal of Ethidium Bromide from DNA by Extraction with Organic Solvents
I did try sci-hub but still didnt find the full text.
So if u can access this please share here Dr. Alim .
and share your view on above idea.
There are several protocol for isolate DNA but not proteins from a complex mix. One of the most used is PEI precipitation. Another may be use a cationic resin matrix like Q-sepharose.
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