Aqueous Phase of DNA extraction - DNa extraction (Dec/18/2005 )
ive been doing some DNA extractions by initially adding phenol to the dna solution, thereafter removing the aqeous phase(1) , adding equal amounts of chloroform/isoamyl(25:1) to that aqeous phase(1) , centrifugating and removing the the second aqueous phase(top layer) from that. Now some of the aqeous phases were clear, however some were rather milky , especially at the phenol stage. Am i correct to think that the top layer of the phenol stage was were the DNA should be. It stands to reason as your phenol layer sits at the bottom, rite. and so does cholorform? That either at the phenol or chloroform stage, after centrifugation all protein and debri will sit at the bottom with the respective solutions while the nucleic acids move to the top layer. The only time during the extraction that DNA will be at the bottom is once you add the ethanol and sodium acetate, and centrifuge(cos at that stage i should see a pellet at the bottom..right??)
So the top layers that i took were infact were the DNA ? but why were they milky , cos i expected them to be clear??
thankyou in advance to anyone that can confirm and answer my dillema
If you have lots of protein, it may be necessary to extract with phenol/chloroform a second or third time to remove the protein. The solution should be clear when protein is removed. Do a final chloroform only extraction to remove residual phenol, then ethanol precipitate.
thankyou phage434, but what happens if you have already gone into the ethanol:acetate precipitation stage? i thought of mayb adding a 1:1 phenol:chloroform mixture once i centrifuged the ethanol:acetate mixture.THe DNA would pellet out, i'd remove the supernatant, and then add the phenol:chlroform mixture(1:1) to that pellet to get rid of the unecessary proteins that got sucked up? but as ethanol and phenol:chlorofrom dissove , i guess that wont work.? any suggestions