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I hope this sort of topic isn't frowned upon here (I read some topics like this a while back), but I'd like to hear from some of you guys on this, since I'm sure you've done this before.
I'm doing RNA extraction from tissue by homogenizing in Trizol (1mL), adding chloroform (200µL), vortex well, spin down, take off top, clear layer (avoiding protein/DNA layers), add 500µL 2-propanol, vortex, spin, aspirate, add 500µL 70% EtOH, vortex, spin, aspirate, resuspend in DEPC-treated H2O.
I can get an OK yield, but my problem is always PURITY (OD260/OD280)! I cannot, for the life of me, get above 1.5 or 1.6, yet I know that I want 1.9+. Do you have any suggestions as to what I might be doing wrong? I know the most important step is not aspirating any of the protein/DNA layer after the first spin. But I'm spinning at 14k RPM for 30 minutes, so it's pretty much as solid as it's going to get, and I'm using a small pipet and pipetting as SLOOOWLY as possible so as not to disturb the layers below. Also, this last time I did it, I even left about 50µL of the clear layer that I could likely have taken off, and still, purity values of 1.5 or 1.6. I know that I'm not the greatest pipetter in the world, but I've had good results with real-time PCR, etc.
I know that you can just do a purification step, but I'd like to try to avoid it. I know lots of people that can get 1.9+ without purification step. Are there any secret tips you guys have?
I don't know if Trizol goes bad either...
I'm doing RNA extraction from tissue by homogenizing in Trizol (1mL), adding chloroform (200µL), vortex well, spin down, take off top, clear layer (avoiding protein/DNA layers), add 500µL 2-propanol, vortex, spin, aspirate, add 500µL 70% EtOH, vortex, spin, aspirate, resuspend in DEPC-treated H2O.
I can get an OK yield, but my problem is always PURITY (OD260/OD280)! I cannot, for the life of me, get above 1.5 or 1.6, yet I know that I want 1.9+. Do you have any suggestions as to what I might be doing wrong? I know the most important step is not aspirating any of the protein/DNA layer after the first spin. But I'm spinning at 14k RPM for 30 minutes, so it's pretty much as solid as it's going to get, and I'm using a small pipet and pipetting as SLOOOWLY as possible so as not to disturb the layers below. Also, this last time I did it, I even left about 50µL of the clear layer that I could likely have taken off, and still, purity values of 1.5 or 1.6. I know that I'm not the greatest pipetter in the world, but I've had good results with real-time PCR, etc.
I know that you can just do a purification step, but I'd like to try to avoid it. I know lots of people that can get 1.9+ without purification step. Are there any secret tips you guys have?
I don't know if Trizol goes bad either...