simple question - (Dec/02/2005 )
I am new in molecular biology and I am so confused by all the DNA/RNA extraction protocol.
Here is some of my question:
1. why there is so many different salt been used in DNA/RNA extraction (KAc, NaAc, NH4Ac,
NaCl.......... ). How much to add? Can I get more DNA/RNA if more salt is added?
Which one is suitable in DNA and which one is for RNA extraction ?
2. I know that isopropanol can precipitate DNA/RNA, but if I have very little nucleic acid, will it be
good if I add more isopropanol, just to ebhance precipitation.
3. Is there such thing like certain salt can only go with Ethanol, certain salt must go for alcohol....?
4. Sometime when I quantitate my RNA using spectrophotometer, my reading is 12.3 ug/ml but
more surprisingly ismy A260:A280 ratio > 3. What does it mean.... very pure ??
I am just so confuse
question 1 : i don't know if it's the type of salt used or the pH used that does the job.
I know that NaAc is to use at 3M and NH4Ac is at 5M but pH is almost the same 5.3 or 5.5. Combination salts + Ph finally do the job
In protocols salts are added in excess so adding more salts will not that help and it will raise the difficulty to remove efficiently salts during 70%EtOH washing step.
Qestion 2 : No
IPrOH is more hydrophobic as ethanol (and butanol is more than IprOH) So it efficiently drive the precipitation of nucleic acids as a 1:1 ratio. (50% IprOH = 70%EthOH, but need Ethanol wash :lol)
Question 3 : I don’t get very well this question as alcohol is generally ethanol.
Salts are pulled down by the relative hydrophobicity.
question 4 : facing RNA purity, ratio should be 2.2 at max
see the topics down :
I think my third question is not relevant anymore
By the way, if suspect my RNA is too little to be precipitated, can I use butanol instate of isopropanol? How much shoud i used?
there are several coprecepitants (check cataloques) that which will help to precipitate even very small amounts
Thank you very much. You had provide me good information.
Those magical stuff in kit so call RNA carrier is just not more then glycogen, acrylamide, tRNA......
We got planty in our lab!!
Can start using now
Buy the way, can anyone explain to me how/why my A260:A280 ratio become >3?
Does is mean VERY PURE or OVERLY PURE??
hm, depends ... does the carrier you now use, absorp at 260/280 ?
I haven't use any just yet. Does carrier RNA contribute to OD reading.
Can I perform an extraction control (carrier + H2O) and use it as my reference od spectrophotometer reading?
hm, i would think, as you'll have to add quite a lot carrier, that most of the absorbance is due to the carrier, which makes this way to quantify rather unreliable. better use something els instead of RNA which is not absorbing at 260
i think t's not the best method... as you may have differences on each sample, and that is a problem for comparison between experiments.
Glycogen is reliable and does not absorb at 260. And it's easy to wash in great volume of "alcohol"
Thank for you guys. By the way, can acrylamide contribute to OD reading?
I got another question. What happen if my add some RNase inhibitor inside my extracted RNA, would it be good to stop RNase from eating up my RNA?