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I don't know if I have RNA pellet or not - There are very little amount of RNA, Is there anything I can see to see the pell (Jun/05/2006 )

I’m extracting viral RNA from allantoidal liquid using trizol to do RT-PCR. There are always very little amount of RNA so I don’t see any pellet after precipitation, but most of the time I get a good band after PCR. The problem is that sometimes, I don’t have any band (my positive control is ok), if I repeat the RNA extraction (and RT-PCR), then I have the band. I’m almost sure that the problem is that sometimes I lose the RNA pellet when I wash with ethanol (75 %) or something like that, but as I never can see the pellet, I don’t know if I’m losing it or not. What can I do? Is there anything I can see to see the pellet??. Please help me!!! sad.gif

-aztecan princess-

after lysis and phase separation, you pipett the upper phase and add 1µl of a 100mg/ml glycogen as a carrier. Then isopropanol. So you'll see the pellet of glycogen, and by the way the RNA is inside.

-fred_33-

QUOTE (fred_33 @ Jun 5 2006, 11:57 AM)
after lysis and phase separation, you pipett the upper phase and add 1µl of a 100mg/ml glycogen as a carrier. Then isopropanol. So you'll see the pellet of glycogen, and by the way the RNA is inside.


Thank you very much... I’ll do it. Can I precipitate with ethanol or isopropanol after add glycogen? Is there any special treatment I have to do after that?
Again thank you very much! biggrin.gif

-aztecan princess-

you add glycogen and vortex few seconds. That ensure homogeneization.
After that point you add the alcohol of your choice and mix.
Then proceed as normal.

-fred_33-