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260/230 ratio


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5 replies to this topic

#1 Telomerase

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Posted 12 October 2009 - 06:39 AM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.
"Beware the power of a PhD student" - scolix

#2 Flyingbike

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Posted 12 October 2009 - 09:30 AM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.



when you precipitate you have to add glycogen and NaAc before ethanol, right ?
Are 3 ethanol washes really necessary ? As you certainly use 70% etoh, some of your RNA may be lost in 30% water.

#3 Telomerase

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Posted 12 October 2009 - 10:20 AM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.



when you precipitate you have to add glycogen and NaAc before ethanol, right ?
Are 3 ethanol washes really necessary ? As you certainly use 70% etoh, some of your RNA may be lost in 30% water.


I'm not sure if removing some of the washes won't make the problem worse.
It's the ratio that worries me more than yield, which won't be much better in these conditions. Some protocols warned me about the need of nested PCRs, and I am doing well without it, so it's ok. The reliability of PCR worries me though, because of ratios.

Edited by Telomerase, 12 October 2009 - 10:22 AM.

"Beware the power of a PhD student" - scolix

#4 Flyingbike

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Posted 12 October 2009 - 10:32 AM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.



when you precipitate you have to add glycogen and NaAc before ethanol, right ?
Are 3 ethanol washes really necessary ? As you certainly use 70% etoh, some of your RNA may be lost in 30% water.


I'm not sure if removing some of the washes won't make the problem worse.
It's the ratio that worries me more than yield, which won't be much better in these conditions. Some protocols warned me about the need of nested PCRs, and I am doing well without it, so it's ok. The reliability of PCR worries me though, because of ratios.


Are you sure there is a link between your 230 ratio and PCR efficiency ?

My modest experience is that 230 ration highly depends on the kind of extraction used. Did you try spin columns for example ?

#5 Telomerase

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Posted 12 October 2009 - 01:03 PM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.



when you precipitate you have to add glycogen and NaAc before ethanol, right ?
Are 3 ethanol washes really necessary ? As you certainly use 70% etoh, some of your RNA may be lost in 30% water.


I'm not sure if removing some of the washes won't make the problem worse.
It's the ratio that worries me more than yield, which won't be much better in these conditions. Some protocols warned me about the need of nested PCRs, and I am doing well without it, so it's ok. The reliability of PCR worries me though, because of ratios.


Are you sure there is a link between your 230 ratio and PCR efficiency ?

My modest experience is that 230 ration highly depends on the kind of extraction used. Did you try spin columns for example ?


Quite sure. Sometimes there is a variation in the ratio, and if it's quite a difference, I see it in the PCR. This bugs the experiment and I have to repeat the whole stuff. I always use Trizol/chloroform. How much do you lose on the columns, comparing to Trizol?

Edited by Telomerase, 12 October 2009 - 01:04 PM.

"Beware the power of a PhD student" - scolix

#6 Flyingbike

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Posted 12 October 2009 - 11:47 PM

Hello all.

This is a variation around the very often asked question.
I coimmunoprecipitate RNAs with my protein, so the yield is usually around 150 ng of RNA. That means, the spectrophotometer barely gets the readout, if I dilute it 10x and I can't afford spending more sample just to measure. The ratios 280/260 are fair enough for such a low yield, around 1.5, but the 260/230 are below 0.5 and I am essentially afraid of doing anything to my sample not to lose the material.
I precipitate the RNA with ethanol, plus additional glycogen and NaAc, in -80 overnight and I do as many as 3 ethanol washes. PCRs work, but I'd like to get the ratio higher, to eliminate differences in the bands because of potential PCR inhibitors.
Of course, the RNA is probably a bit degraded after a whole co-ip procedure, so does that add to the total? Increasing the yield is not really an option - I'd have to spend fortune on the beads.



when you precipitate you have to add glycogen and NaAc before ethanol, right ?
Are 3 ethanol washes really necessary ? As you certainly use 70% etoh, some of your RNA may be lost in 30% water.


I'm not sure if removing some of the washes won't make the problem worse.
It's the ratio that worries me more than yield, which won't be much better in these conditions. Some protocols warned me about the need of nested PCRs, and I am doing well without it, so it's ok. The reliability of PCR worries me though, because of ratios.


Are you sure there is a link between your 230 ratio and PCR efficiency ?

My modest experience is that 230 ration highly depends on the kind of extraction used. Did you try spin columns for example ?


Quite sure. Sometimes there is a variation in the ratio, and if it's quite a difference, I see it in the PCR. This bugs the experiment and I have to repeat the whole stuff. I always use Trizol/chloroform. How much do you lose on the columns, comparing to Trizol?


I don't know, but quantity of RNA that can bind to column is limited. I don't think it is a problem for you. The thing is that I obtain a peak at 230 nm when I use an equivalent of trizol, which disappears with columns. Ive never been able to explain this peak, but Ive never found it to interfere with downstream reactions.




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