transfection with AS oligos ~ control? - (Jul/04/2005 )
Hi,
If you were going to transfect some cells with AS oligos, how would ensure they actually make it into the cells? GFP? label the oligos?
Cheers,
vetticus
Hi vetticus,
Just curious why don't use siRNA. In siRNA transfection, we don't usually measure how much siRNA enters the cells, but measure the effect it may cause. Certainly you can label your oligos.
Just curious why don't use siRNA. In siRNA transfection, we don't usually measure how much siRNA enters the cells, but measure the effect it may cause. Certainly you can label your oligos.
because i'm not the one who thought up this experiment. i was told: "here are some oligos, go do stuff with 'em."
ok.labelling oligos, any good protocols out there?
Ambion sells a kit for RNA oligo labeling. it is called silencer siRNA labeling kit and can be used for ssRNA labeling.
Thanks!!!
hey you can label the oligos with p32 and then measure the radioactivity incorporated within the cells. The amount of radioactivity will correspond to the amount of the oligo that has been able to slip into the cell.
Lemme know if you need more details and if u can work with radioactivity. I can give you the details. The procedure is very simple
p..
PS why is this post appearing as "new post" when it appears to be posted initilaly in 2004???
Lemme know if you need more details and if u can work with radioactivity. I can give you the details. The procedure is very simple
p..
PS why is this post appearing as "new post" when it appears to be posted initilaly in 2004???
definately need more details... and can work with radioactivity (yay). Thanks!
Ps, 2004? a time warp maybe. someone call steve hawkins.
hi
forn labelling oligos, i use T4PNK and ATP gamma-32P at 6000Ci/mmol with a molar ration between ATP and the molecule to label at 1:1.
1h at 37°; i've noticed that heating the oligos at 65° 10' and chill on ice for 10' before the label enhances it.