puzzling RT question - (Jul/26/2007 )
Hi everyone,
I've been trying to get a first-strand cDNA synthesis to work, but have been having no such luck so far. I'm using SS III with the invitrogen kit. In most of my samples, I get about the same cDNA concentration back with the RT samples and no RT added controls. My water controls (where no RNA template is added), I get back concentration ranging from 300-500 ng/microliter. Can this indicate a problem with the dNTP mix or the oligo(dT)? I would appreciate any input. Thanks!
how's the bulb on your spec?
I'm only trying to do a first-strand cDNA synthesis with no amplification from about 600 ng of starting RNA. Upon synthesis, I precipitate the cDNA and check the concentration but there always seems to be false positives! The invitrogen protocol works between 10 pg - 5 micrograms of total RNA so I'm not sure what goes wrong...
that's why I suggested checking your spec
if all else is exhausted, it's important to verify that your spectrophotometer is working properly
first, though, how do you ppt your cDNA? perhaps there is some residual stuff in there that's giving you your false-positive reading. what are your ratios?
if all else is exhausted, it's important to verify that your spectrophotometer is working properly
first, though, how do you ppt your cDNA? perhaps there is some residual stuff in there that's giving you your false-positive reading. what are your ratios?
I'm pretty sure the spec is working properly...
I ppt by adding 10% of total volume 3M NaOAc pH 5.0 and 3 Volume of absolute ethanol stored at -20. There's definitely something else being ppt'ed...