problem with phase separation using Tri-reagent - (Nov/03/2008 )
Hi,
I am now facing with the problem of high genomic DNA contamination in RNA extraction using Tri-reagent. I am suspecting the co-extraction of DNA during phase separation. During phase separation, it is said to hv RNA in upper aqueous phase, DNA in interphase, and protein in lower red organic phase. How could I differentiate between the interphase and aqueous phase? both are jst colorless. Thanks!!
Ambion TriReagent is red at and separates after adding chlorophorm to a colorless uper aquaeous phase, some garbage in the middle, and lower red organic phase.
Is you don't see this clearly after centrifugation, then there is something wrong with your TriReagent.
DNA is indeed separated too, most of it is the "garbage" you see in the interphase, but some can be in the lower 1/3 of aquaeous phase. That's why we only take the upper 2/3 of it. Still to be sure, we use Ambion DNAfree to get rid of the rest.
Is you don't see this clearly after centrifugation, then there is something wrong with your TriReagent.
DNA is indeed separated too, most of it is the "garbage" you see in the interphase, but some can be in the lower 1/3 of aquaeous phase. That's why we only take the upper 2/3 of it. Still to be sure, we use Ambion DNAfree to get rid of the rest.
Hi, I am dealing with bacterial cell suspension, shld I use Tri-reagent LS (liquid sample instead)? previously, I used the normal Tri-reagent and I really didn't get to see anything in the interphase. Thanks!
I don't thing you need to. Just spin your culture and add TriReagent to the pelet of bacterial cells. TriReagent LS is for samples like blood and so, it's only a more concentrated TriReagent.
If you routinely need to isolate DNA only, then maybe TriReagent is not a best choice. Use classic phenol/chlorophorm method or DNA kits. TriReagent is handy when you need RNA, or both RNA, DNA or even proteins isolated from one sample. That's why it's called TriReagent.