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RNA contamination w/ ethanol - (Jan/13/2006 )

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Hi, Im new here so my question might have been already answered somewhere eslse....
I think my RNA is contaminated with ethanol (75%) cuz i dont think it dried completly before resuspending RNA with Rnase free water. I know this because for A260 I got -2.108 and for A280 I got -1.766 (notice negative #s). Does anyone know how RNA interacts with ethanol if frozen at -80 C for 4 days? If no significant interaction, how can I uncontaminate it, or get rid of ethanol? Should I add isopropanol again, etc ... then centrifuge 12000x g for 10min at 4C, remove supertanant and then wash RNA with ethanol again? Thanks in advance!

-rana-

u should read through Molecular Cloning (A laboratory manual by Sambrook and Russell) book

to get any information related to DNA and RNA work.
thx







QUOTE (rana @ Jan 13 2006, 06:46 PM)
Hi, Im new here so my question might have been already answered somewhere eslse....
I think my RNA is contaminated with ethanol (75%) cuz i dont think it dried completly before resuspending RNA with Rnase free water. I know this because for A260 I got -2.108 and for A280 I got -1.766 (notice negative #s). Does anyone know how RNA interacts with ethanol if frozen at -80 C for 4 days? If no significant interaction, how can I uncontaminate it, or get rid of ethanol? Should I add isopropanol again, etc ... then centrifuge 12000x g for 10min at 4C, remove supertanant and then wash RNA with ethanol again? Thanks in advance!

-hasina-

[quote name='hasina' date='Jan 13 2006, 08:11 PM' post='37021']
u should read through Molecular Cloning (A laboratory manual by Sambrook and Russell) book

to get any information related to DNA and RNA work.
thx

thanks hasina... i found a 1982 edition at my university library website, do u think its too old? i think i only need to read chapter 7, do u know if i can find it online?

-rana-

I already posted this in General lab tech. but I think I'll get more replies here. hasina recommended I read "Molecular Cloning (A laboratory manual by Sambrook and Russell)", but that might take sometime before I even get the book... heres the question:

"Hi, Im new here so my question might have been already answered somewhere eslse....
I think my RNA is contaminated with ethanol (75%) cuz i dont think it dried completly before resuspending RNA with Rnase free water. I know this because for A260 I got -2.108 and for A280 I got -1.766 (notice negative #s). Does anyone know how RNA interacts with ethanol if frozen at -80 C for 4 days? If no significant interaction, how can I uncontaminate it, or get rid of ethanol? Should I add isopropanol again, etc ... then centrifuge 12000x g for 10min at 4C, remove supertanant and then wash RNA with ethanol again? Thanks in advance!"

-rana-

um, yeah, his suggestion was very good if you can get your hands on a copy. even though it is old, it is still the bible...

another excellent resource for RNA work is www.ambion.com; they have great technical literature and they are the RNA people

I would say your RNA should be fine if there has been no contamination with RNAse (by far your biggest worry) I would think you can repeat the precipitation (I usually do ethanol/acetate with my RNA but there are multiple methods) and re-spin the pellets; whatever you do make sure to dry them thoroughly before resuspension...and please be careful, your pellets will probably be soft when you go to remove the supernatant


if there is any more doubt, you may run a gel to check them; that can be helpful


good luck

-aimikins-

thanks aimikins, it wouldnt hurt to do precipitation again and run the gell...

QUOTE (aimikins @ Jan 14 2006, 10:08 AM)
um, yeah, his suggestion was very good if you can get your hands on a copy. even though it is old, it is still the bible...

another excellent resource for RNA work is www.ambion.com; they have great technical literature and they are the RNA people

I would say your RNA should be fine if there has been no contamination with RNAse (by far your biggest worry) I would think you can repeat the precipitation (I usually do ethanol/acetate with my RNA but there are multiple methods) and re-spin the pellets; whatever you do make sure to dry them thoroughly before resuspension...and please be careful, your pellets will probably be soft when you go to remove the supernatant


if there is any more doubt, you may run a gel to check them; that can be helpful


good luck

-rana-

Using isopropanol could be worse than ethanol actually, because it is more difficult to get rid of by drying.

-sergechampetier-

QUOTE (sergechampetier @ Jan 16 2006, 06:53 AM)
Using isopropanol could be worse than ethanol actually, because it is more difficult to get rid of by drying.


im actually still using ethanol to wash RNA with in the last step. But before that, this is the what im doing:

-add trizol, incubate...add choloform, shake, incubate, centrifuge, seperate phenol-chlofoform layer and aqueous (with RNA) layers.

-add ISOPROPANOL to aqueous layer,incubate,centrifuge...remove supernatant, wash RNA w/ 75% ethanol,vortex,centrifuge, decant & air dry.

do u mean for the last step u use isopropanol? any other recommendations?
thanks biggrin.gif

-rana-

QUOTE (rana @ Jan 16 2006, 10:52 AM)
do u mean for the last step u use isopropanol? any other recommendations?
thanks biggrin.gif

I meant NOT to use isopropanol for the last step. But the way you do seems OK to me.
I am more concerned by your negative ODs. I don't know if ethanol can really cause this, but my best guess would be to have another RNA you can trust (e.g. from commercial source), just to make sure nothing is wrong with your spectro instrument.
You can also roughly estimate the amount of ethanol remaining (no more than than a couple of microlitre, certainly) and add it on purpose to some RNA in order, to see if ethanol is the right explanation for your problem.
Good luck.

-sergechampetier-

You need to get rid of any ethanol before you resuspend your pellet as residual alcohol will seriously inhibit your downstream processes

I leave my tubes inverted on a tissue (after physically removing as much ethanol as possible) to air dry for 30 mins, and check for ethanol by smell before resuspension

-John Buckels-

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