Regarding RNA extraction - darn pellet (Jul/07/2008 )
I'm currently in the process of extracting RNA. I've treated my samples with sodium acetate, saturated phenol, chloroform isoamy alcohol, phenol chloroform isoamyl alcohol, centrifuged at 13000 for 3 minutes. I removed supernatant and transferred to new tubes and I added isopropanol and centrifueged again at 16,000 for 20 minutes. At this point, I'm supposed to remove the supernatant and leave the pellet at the bottom. However, the pellet always resuspends regardless of how careful I am when pipetting. Have any of you encountered this difficulty at all? I've found that I can sometime preserve the pellet by pipetting it against the walls of the tube and quickly removing supernatant.
-Christian Ortiz-
QUOTE (Christian Ortiz @ Jul 7 2008, 07:10 AM)
I'm currently in the process of extracting RNA. I've treated my samples with sodium acetate, saturated phenol, chloroform isoamy alcohol, phenol chloroform isoamyl alcohol, centrifuged at 13000 for 3 minutes. I removed supernatant and transferred to new tubes and I added isopropanol and centrifueged again at 16,000 for 20 minutes. At this point, I'm supposed to remove the supernatant and leave the pellet at the bottom. However, the pellet always resuspends regardless of how careful I am when pipetting. Have any of you encountered this difficulty at all? I've found that I can sometime preserve the pellet by pipetting it against the walls of the tube and quickly removing supernatant.
Isopropanol pellets are loose and more transparent, always a headache.
1. Use Glycoblue, to easily locate it.
2. Take out most of the sup, leave behind 20-50 ul, brief spin it again.
3. mark the eppendorf wall with a marker where pellets are likely to situate.
-cellcounter-