The Basics by huangyuarong


									                 The Basics: How Ethanol
                 Precipitation of DNA and RNA                                                        Tech Tips

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About the author Tag this

                Nick Oswald

Nick is a molecular biologist-turned-publisher. After a PhD in Developmental Biology and an eclectic seven years in biotech he is now Editorial
Manager ofNeuroendocrinology and the founder and Editor-In-Chief of Bitesize Bio. You are welcome to connect with Nick onLinkedIn

Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid (DNA or RNA)
preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous
solution, which forces the nucleic acid to precipitate out of solution. The precipitated nucleic acid can then be
separated from the rest of the solution by centrifugation. The pellet is washed in cold 70% ethanol then after a
further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being
resuspended in clean aqueous buffer. So how does this work?

A bit about solubility…

First we need to know why nucleic acids are soluble in water. Water is a
polar molecule – it has a partial negative charge near the oxygen atom
due the unshared pairs of electrons, and partial positive charges near the
hydrogen atoms (see the diagram on the right).

Because of these charges, polar molecules, like DNA or RNA, can
interact electrostatically with the water molecules, allowing them to easily
dissolve in water. Polar molecules can therefore be described as
hydrophilic and non-polar molecules, which can’t easily interact with water
molecules, are hydrophobic. Nucleic acids are hydrophilic due to the
negatively charged phosphate (PO3-) groups along the sugar phosphate backbone.

The role of the salt…

Ok, so back to the protocol. The role of the salt in the protocol is to neutralize the charges on the sugar
phosphate backbone. A commonly used salt is sodium acetate. In solution, sodium acetate breaks up into Na+
and [CH3COO]-. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the
nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water.

The role of the ethanol…

The electrostatic attraction between the Na+ ions in solution and the PO3- ions are dictated by Coulomb’s Law,
which is affected by the dielectric constant of the solution. Water has a high dielectric constant, which makes it
fairly difficult for the Na+ and PO3- to come together. Ethanol on the other hand has a much lower dielectric
constant, making it much easier for Na+ to interact with the PO3-, shield it’s charge and make the nucleic acid
less hydrophilic, causing it to drop out of solution.

The role of temperature…

Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (e.g. -20 or -80C) is commonly cited in
protocols as necessary in protocols. However, according to Maniatis et al(Molecular Cloning, A Laboratory
Manual 2nd Edition… 2nd edition?? – I need to get a newer version!), this is not required, as nucleic acids at
concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient.

The wash step with 70% ethanol…

This step is to wash any residual salt away from the pelleted DNA.
A few tips on nucleic acid precipitation…

        Choice of salt
           Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations
           Use Sodium chloride (0,2M final conc) for DNA samples containing SDS since NaCl keeps SDS
              soluble in 70% ethanol so it won’t precipitate with the DNA.
           Use Lithium Chloride (0.8M final conc) for RNA. This is because 2.5-3 volumes of ethanol should
              be used for RNA precipitation and LiCl is more soluble in ethanol than NaAc so will not precipitate,
              but beware – chloride ions will inhibit protein synthesis and DNA polymerase so LiCl is no good for
              RNA preps for in vitro translation or reverse transcription. In these cases, use NaAc.
           Use Ammonium acetate (2M final conc) for the removal of dNTPs, but do not use for preparation of
              DNA for T4 polynucleotide kinase reactions as ammonium ions inhibit the enzyme.
        To increase the yield in precipitations of low concentration or small nucleic acid pieces (less than
         100 nucleotides)
           Add MgCl2 to a final concentration of 0.01M
           Increase the time of incubation ice before centrifugation to 1 hour.
If you have anything to add, please feel free to leave a comment!


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      103 comments on this article already!
                                                                                                                add your comment


                 2 years ago
2.    Great post – more please!
      How phenol extraction works!
      How CsCl centrifugation works!
      How HPLC works!


                 2 years ago
4.    Thanks for the suggestions – I’ll definitely write these articles in the future. Suggestions are always welcome!!


                 2 years ago
6.    Thanks for this article. I have extracted DNA hundreds of times but wasn’t 100% sure how and why it worked. Thanks
      for the explanation!


                 2 years ago
8.    I’m a little bit freaked out by the timing of this post since I was just thinking about this very topic yesterday… Oh
      well, perfect timing. Thank you very much.


                 2 years ago
10. How does the ethanol wash the salt away?


                 2 years ago
12. Arag,

13. Thanks for your question.

14. The wash step removes the relatively small amount of salt that pellets with the DNA. This small amount of salt can
    dissolve in the 70% ethanol/30% water mix – mainly in the water part as the salt is far more soluble in water than in

               2 years ago
16. Is there optimal amount for Ethanol in precipitation? I heard people say more Ethanol you put in the solution, more
    precipitation you will get. But I always have to centrifuge SEVERAL times at 150000 rpm! Every time I centrifuge, I
    get more pellet out! I centrifuge 35min after freezing over night, then remove the ethanol to a new tube. After 5min
    in room temperature I centrifuge 30min again, then even more products can come out! What can be reason! I put 4.5
    V Ethanol. Can that be the reason?


               Shukti Chakravarti
               2 years ago
18. I am training a whole bunch of new people in my lab now and this little article just hit the spot. Thank you.


               2 years ago
20. Very nice site you have. I’m going to subscribe to the RSS. Anyway, I still don’t understand why we need cold ethanol
    at all. Why not at RT?

21. Thanks!


               2 years ago
23. @Ying

24. I;m pretty sure the optimum is 70% ethanol. As I understand it you need some water in there so that salts don’t
    precipitate with the DNA.

25. There are always losses associated with EtOH precipitation, but I think it is the still-solubilized DNA that was the
    reason for the losses.

26. I suppose that the longer you centrifuge the more you would get out, up to a point, but I don’t think the reason you
    are seeing this is the excess ethanol.


               2 years ago
28. @Anne

29. Glad you like the site. The whole procedure is based upon making the DNA insoluble so it precipitates out of solution
    and can be pelleted.

30. The lower the temperature, the less soluble things are generally, so the low temperature helps to make the DNA


               2 years ago
32. is it possible to co-precipitate salt into the sample if we spin the sample at higher speed or for longer period?


               2 years ago
34. Hi Cookie

35. I don’t think so. The salt will only precipitate if there is insufficient water for it to solubilise in. If it is in solution, no
    amount of centrifugation will pellet it.


                amy christianson
                2 years ago
37. cool site


                Jim H
                2 years ago
39. Cookie,

40. Just do the wash in punctilious or 95% EtOH or skip the 70% rinse altogether. The salts aren’t soluble in 95% EtOH and
    will precipitate.

41. I worked with Jim Hartley & Jil Zeugin in the 80′s at BRL (now know as Invitrogen but really not the same). They
    published a study in 1982,which is still disputed by some cement heads, proving that the amount of time in the
    centrifuge is the primary determinant of yield and that cooling the Ethanol actually reduces yield and that incubation
    has no effect on yield.

42. So put your tube right into the ‘fuge after adding your RT EtOH and spin an extra 10 minutes (instead of having it sit
    on ice) and I promise your yield will increase.

43. i have been trying to find the Zeugin & Hartley reference, but since it was published in our trade journal (FOCUS)I
    can’t find it on the web.


                2 years ago
45. Thanks for the explanation – very useful. Why is it that sometimes the DNA just won’t precipitate out at all? I had this
    problem today. I tried cooling and even adding glycogen – nothing!


                2 years ago
47. Any sugestion how increase amount of DNA. Precipitation was done after reaction with BigDye 3.1. Fragment length
    80 – 150 bp.


                2 years ago
49. wonderful!
      how many experienced people in a lab wouldn’t be able to explain it that well


                2 years ago
51. thanx for the information,but i want to know if peg is having any effect on rna precipitation


              2 years ago
53. What is the ‘dynamic range’ of precipitation in term of fragment length? That is, do small fragments and nucleotides
    precipitate as well?


              Jim H
              2 years ago
55. shhigg,

56. The “dynamic range” of ethanol precipitation is in the range of 18-20 nucleotides, depending on their sequence. Over
    20 bp and they will precipitate.

57. Free, non-bound nucleotides will not precipitate under standard conditions.

58. Smaller length oligos, RNA or DNA fragments are purified using one of the many commercially available “spin
    columns” or some other solid matrix, like biogel (per maniatis pp 11.34-11.37)


              2 years ago
60. I have question. I have been isolating BACs in 96 well blocks for a while now and I do not get consistently good
    results. The results I hope to achieve are to have clear samples of DNA dissolved in water. However, I often get white
    junk in my well at the end that will not dissolve. The white junk appears after I transfer lysate from the wells (after I
    have added a cell lysis solution of NaOH and SDS then finally adding potassium acetate and leaving on ice for 20mins)
    into 300ul of isopropanol to precipitate the DNA. I sometimes let it precipitate at room temp for 20mins or
    sometimes at -80 for 15 (which is better?)before spinning it at 4000rpm for 30 minutes. The last step is washing with
    70% etoh . I dont understand why there is white stuff in my wells that do not dissolve after I dry off the the ethanol
    and water. can you tell me which step could be affecting these results.


              2 years ago
62. Thanks for good explaination.

63. I’ve got one question. Why do we sometime also expected to add Sodium EDTA together with Sodium Acetate for


              2 years ago
65. @Ali: It sounds like the white junk is the cell debris and the reason is a problem with the centrifugation step.
    4000rpm is too slow to separate the supernatant from the white debris so with these centrifugation parameters you
    will always have some of the debris carry over into the supernatant. Centrifuging at 10,000 rpm+ for 15-30 minutes
    should help.

66. @Dana: Sounds to me like the sodium ions will help with the charge neutralisation of the phosphate backbone and the
    EDTA will chelate any available Mg2+, preventing nucleases from damaging the DNA after precipitation.


              2 years ago
68. Hi,

69. I just heard that DNA will not precipitate if the Ethanol/isopropanol is added before the salt. Is this true? By accident
    I did this last week and I still retrieve much of my DNA. I do leave the tubes overnight in the -20C. So did I retreive
    my DNA or something else?

70. Thanks for your help

              2 years ago
72. Hi Magy

73. The order in which the ethanol and salt are added will make no difference so it sounds like you got your DNA,
    although you’d be best to check on a spec and/or a gel to make sure!

74. Nick.


              2 years ago
76. Thanks for this very nice website. It’s quite helpful.
    1. About the “final conc” of the salt, does it mean the conc in the solution including ethanol (or before adding
    ethanol)? What would happen if the conc of salt is too high?
    2. In some protocols, people use 2V of ethanol for DNA precipitation. What would be the optimal ratio?


              1 year ago
78. In precipitation procedure,I got white cloudy soln after adding chiled ethanol?Why it happens?


              1 year ago
80. Why do I need less isopropanol (1V) if compared to ethanol (3V) when precipitating DNA/RNA? Why is it not necessary
    to add salt when using isopropanol? How can I justify it in chemucal terms?


              1 year ago
82. I am currently trying to run PCR on some samples that underwent EtOH precip. protocol over a year ago. The samples
    were stored at -70C and when i took them out of the freezer they were very cloudy. After doing PCR and running a
    gel I received product for my control, but did not receive product for any of the samples that were cloudy. I was
    considering running the EtOH precip. again, but have limited sample material. Any idea why these sample are cloudy
    and/or what can be done with them?


              1 year ago
84. @CL: The final conc means the concentration after everything has been added, including the ethanol. 2 volumes of
    ethanol is added to give a final concentration of 70% ethanol. I believe this is optimal, but I am not 100% sure.

85. @Paula: Less isopropanol is a less polar solvent than ethanol, so you get the same effect as ethanol but with a lower
    concentration of solvent.

86. @Sarah and anand: The most likely reason for the white precipitate is contamination with protein. You could try
    cleaning up the solution with no loss of product using something like Stratagene’s Strataclean resin.


              1 year ago
88. Thank you for this website. I just have one question: how does the 70% ethanol wash the pellet at the end?

                  1 year ago
90. @Yara. The salt is very soluble in water, so will easily dissolve in the water in the 70% ethanol


                  1 year ago
92. but why doesn’t the DNA dissolve as well?


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       1 year ago

                  1 year ago
95. why do v have to incubate at -80 after adding ethanol..generally temp is reduced to decrease the movement of
    molecules..wht is it doing here??
    this is an awessssome site!!!!!!!!!!


                  1 year ago
97. Here is a suggestion to increase the recovery yield especially for very low amount: You can add linear acrylamide
    before adding Ethanol at a concentration of 5 or 10 µg/ml.

98. Other carrier as been used in the past such as glycogene. Acrylamide is better since no absorption is observed at 260
    nm (which is not the case for most glycogene preps)

99. Thanks for the site it is very helpfull


                  1 year ago
101. Thanks for the explanation~
     I have to extract RNA from skeletal muscle but the 260/230 ratios are not consistenly good (260/230 ~ 1.8-2.0 is
     good? for mine it’s ususlly 0.9-1.3). Is there a way to improve this?


                  1 year ago
103. Thanks! You’re explanation helped me a lot!

104. Cool site!


                  1 year ago
106. Thanks for the explanation. But what about ISOLATION OF YEAST RNA? What makes yeast an ideal source for RNA

               1 year ago
108. @Yara. I should explain it the other way around : the DNA does not dissolve because it is poorly soluble in 70%
     ethanol but the salt is soluble enough in 70% ethanol to dissolve, so the DNA and salt are separated.

109. @Ritu. The idea is that for most solvents, including ethanol, the solubility (i.e. the amount of solute they can hold)
     decreases with temperature. So by lowering the temperature, you theoretically lower the amount of DNA that the
     ethanol can hold in solution. (see But according to Jim (see
     comment near the top of this list), ethanol precipitation works just as well at room temperature as -20/80.

110. @Cyril – Thanks – that is useful

111. @Ange – It might be the kit you are using. I find that the ratios are bad for certain kits and nothing seems to improve
     it. But if the RNA you have isolated is doing the job without any problem then there is no problem… just because the
     ratio is bad does not mean that the sample is no good, it is just one indicator of quality.

112. @Fe – I suppose yeast is only an ideal source for RNA extraction if you want to do something with yeast RNA. If you
     mean why is yeast used for commercial preps of RNA used for (e.g.) blocking the answer is probably that yeast is a
     relatively cheap source of RNA – economical to grow and relatively easy to break open and isolate the RNA.


               1 year ago
114. Need help!!

115. Recently I tried to purify RNA (46 bases) with 20% denaturing PAGE. After finishing the gel running, I dyed the gel
     with EB and then cut the band of RNA under UV. Then I put the gel slice into elution buffer (0.5 M NH4OAC, 0.1M
     EDTA, 0.1% SDS) at room temperature over night and final volume is about six ml. After that, I did butonol extraction
     experiment and a final solution of 0.4 ml was obtained. 1 ml Ethonal was then added. The tube was buried in dry ice
     for two hours and much white precipitate was observed. I centrifugated the tube 30 min and got a large amount of
     precipitate and at the same time, the upper supernatant contains two layers. I removed the supernatant and used
     70% ethonal (in DPFC water) to rinse the precipitate twice in order to remove the salt components. Then I dried the
     precipitate left and made a solution of about 0.4 ml with DPFC water. However, the OD measured is about 0.072 (the
     crude RNA used is 6 OD). It seems that there is no RNA in the final product.

116. What is the problem with my procedure?
     How to purify RNA with length below 100 bases by native PAGE?

117. Thanks a lot!


               1 year ago
119. i am exprience an unussual problem. ETOH precipitation after restriction with VSP1 resulted in an additional band
     that was not seen in a gel before the precipitation. The water used for 70% etoh preparation and the TE used to
     dissolve the DNA were checked for contamination and were found negative. Any idea someone? Can etoh triger an
     unspecific resriction?


               Jim H
               1 year ago
121. Avi,

122. You don’t give enough information about the DNA you’re cutting, but I wouldn’t suspect your EtOH or water.

123. One possibility, especially if you’re doing a restriction digest of a plasmid, is that you’re seeing both the linear and
     supercoiled form of the plasmid.

124. Typically, “contamination” with non-specific nuclease will just give you a smear and not a distinct band on a gel.


               1 year ago
126. Hi,

127. I’m having a similar probelm cleaning my plasmid digests. After digestion, I phenol:chloroform extract the DNA and
     then extract once more with chloroform to ensure removal of trace phenol. At this point, the digest looks the same
     as before. THE PROBLEM is after I ethanol precipitate, there is a ton of streaking and the larger bands are sometimes
     wholly absent. Am I seeing DNAse contamination?


              Paul M
              1 year ago
129. Streaking maybe due to too vigorous pipetting or vortexing, especially if DNA is long. It can also be due to overdrying
     of DNA after precipitation.


              Paul M
              1 year ago
131. After looking for quite some time I finally located two good articles about ethanol precipitation, one of them is the
     one Jim H. was talking about, they are available in archive on invitrogen website.

132. Hmmm, it seems I can’t post the links here I added them to wikipedia article on Ethanol Precipitation so just google
     it and look at the bottom of the page on wiki.
     I also added links to them to wikipedia article on ethanol precipitation.

133. Zeugin JA, Hartley JL (1985). “Ethanol Precipitation of DNA”. Focus 7 (4): 1–2.
     Crouse J, Amorese D (1987). “Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate”. Focus
     9 (2): 3–5.


              Kay Jones
              1 year ago
135. After doing ethanol precipition, I put my DNA in the -20 freezer so that I can run PCR the next day. Am I supposed to
     centrifuge my DNA sample again before I run the PCR? if so, for how long?


              Narender Kumar
              1 year ago
137. hey i want to know why only the dna containing part floureses after staining the gel with EtBr ? why not hte whole
     gel flourese ?wat change Etbr undergoes which makes it flourese


              1 year ago
139. Hi,
     I want to know about the required amount of salt to precipitate DNA. in my case, to precipitate DNA 4ug/50ul,
     I added 3M sodium hydroxide 5.5ul and 7.5M ammonium acetate 1/3 Volume. After that, I added 2.5 Volume of EtOH.
     But if add 7.5M ammonium acetate 50ul, is it ok to precipitate this DNA??? Please, answer to me.


              1 year ago
141. Phosphate is PO4 3-

142. Should it not be PO4 3- everywhere you have written PO3-?

143. Thanks

               1 year ago
145. Ok, Just a basic question…at school in my lab we were required to keep the ethanol at a low temperature before
     using it and my SI said it was due to evaporation although she seemed like she was just taking a crack at it rather
     than actually giving me a legit answer.

146. So,
     1. Why is the ethanol kept at a low temperature rather than at room temperature?

147. 2. Would freezing the sample (a calf thymus, not a plant)affect the ability to extract the DNA from the cells?


               1 year ago
149. Jason,

150. The ethanol is kept at a low temperature to reduce the solubility of the DNA so that it precipitates out more
     effectively. Freezing the thymus sample might actually make it easier to remove the DNA by breaking up the tissue a
     bit so that homogenization is easier.

151. Hope that answers your question.

152. Nick


               1 year ago
154. Thanks, this info is awesome.


               1 year ago
156. Hi Nick

157. I have a question. I am trying to precipitate DNA using the commonly used ETOH method. Initially I added 0.1 vol of
     NaOAc (3M pH 5.2)and 2.5 volume EtOH (100%)and I left it for the weekend in the -20. However, I can see that the
     salt has precipitated to the bottom forming a lot of white precipitates! (obviously it is not DNA) and I am worry that I
     have lost my DNA sample as it is really precious from a colon biopsy. Could you tell me if there is anything I can do.


               1 year ago
159. I have a question. In our PCR standardisation we used ethanol precipitation to reduce the concentration of non
     specific product. We stored the PCR product and 100% ethanol mix overnight at -20. Could you tell me if this will
     reduce the concentration of non specific product or do you suggest any changes? Thank you


               YZ Ooi
               1 year ago
161. er… as mentioned, adding salt (natrium acetate)is to neutralize the charge of the sugar phosphate backbond. May i
     1) If total quatity of aqueous solution i had is 800µl. How much of salt and ethanol(95%) i need to add in?
     (normal procedure is 0.1 volume of salt with 2.5-3X volume of 95% alcohol, right?)
     2) If i had added 1 volume of salt accidentally and just added 1volume of alcohol? what will happened?
     3)Any countermeasurement for that situation?

               1 year ago
163. What would happen if you added sodium acetate to a final concentration greater than 0.3 M? Would that affect my
     RNA even though I perform a wash.

164. The story behind the question”
     I am doing a phenol chloform extraction followed by ethanol precipiation.
     I decided to increase my reaction volume by adding salt so i would not have to do this later in my ethanol
     precipiation. That way I increased the volume in which to do my phenol extraction.
     I then added 100% ethanol and pelleted my RNA.


               Preetam Raj
               1 year ago
166. Hello Nick
     Nice to read your description. Easy to know the concept.
     Thank you very much


               1 year ago
168. Two stupid questions:
     1. How to prepare 70% ethanol for DNA or RNA purification?
     2. How is it important that 100% and 70% ethanol must be cold during DNA or RNA purification?


               1 year ago
170. Hello,

171. Your note is very clear and interesing.
     I think Very impotrant is pH of Sodium acetate !

172. For prepare 1 litre of 70 graduate ethanol you have to mix 665g of EtOH 96% and 335g of H2O.


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       10 months ago

               10 months ago
175. Very nice, the explanation is good, but leaves things a little ambiguous… for us students – could you add a
     thermodynamic point of view? It would make everything so much easier to understand…


               10 months ago
177. For some special purpose, I run my DNA sample on a regular agarose gel, then cut the bands. I use 4.7M (final conc)
     NaI and 42C incubation to melt the agarose gel block with DNA. At this step, can (and how) I use ethanol to
     precipitate the DNA? Since there are already plenty salts (NaI at 4.7 M), do I still need to add NaOac as usual? If I put
     this DNA-gel-ethanol in low temperature, can the gel re-solidify and then precipitate with DNA?

               9 months ago
179. Nothing to add, but great info!


               9 months ago
181. Sir you explain all things right no doubt,but i have a query that why we use only Chilled(Ethanol or Isopropanol)i.e
     whats the need for chilling?
     I am very thankful to you if you help me out in this.
     Ohh i get the ans as Jason asked the same question to You.
     Thanx alot sir,I am very thankful to you.
     Plz keep it up…


               8 months ago
183. hi nick,
     i am isolating RNA from a plant sample. i use isoprapanol for precipitation, But everytime i got a lot of contamination
     of polyphenols with RNA. Can u help me to get pure RNA??


               8 months ago
185. Hi Nick,
     I would like to ask you about dna rna precipitation: why dna stays at organic phase at acid ph and rna at aqueous


               8 months ago
187. Every time I do an ethanol precipitation of DNA, I find white flakey precipitate in my sample after resuspending in
     either ddH2O or Tris pH 8.5. This happens regardless of how well I try to wash the pellet with 70% room temperature
     ethanol. Does this happen to anyone else? Is it just left over salt that is no longer soluble for whatever reason? Does
     it affect downstream steps? It doesn’t appear to affect transfection, but I am wondering about enzymatic reactions
     where the salt concentrations matter (e.g. ligation).


               8 months ago
189. Hi John,
     Are you using a silica column method for purification? Or an anion-exchange column (such as qiagen plasmid kits)?
     Sometimes the resin leaks through into the final samples. If this is not affecting any results, you can spin it out and
     transfer the DNA to a new tube.

190. Best,


               8 months ago
192. Hi Suzanne,
193. Thanks for your reply. I do the ethanol precipitation after extracting the DNA from agarose with a Qiagen kit. I
     always like to do an ethanol precipitation after gel purification to remove any excess salts or left over agarose. I find
     this helps with downstream ligation. I only see the white precipitate after the ethanol precipitation, not after eluting
     the DNA off the column. I suppose its possible that the ethanol precipitation knocks the column “leakage” out of
     solution and it remains insoluble afterwards. Is this plausible?


               8 months ago
195. Hi John,
     It is possible. I recall hearing this from other users when I worked at Qiagen (that after ethanol ppt. it appears). If
     you give them a call and ask about it, they might be able to confirm that it is indeed just a little resin and to remove
     it, or if it is something else.
     If the DNA is performing fine in reactions, it is probably just resin.

196. Best,


               8 months ago
198. What is the physical different between precipitation of RNA from precipitation of DNA using ethanol


               Amrita Sinha
               7 months ago
200. Hello Sir,
     I wanted to ask you that why it is so that some DNA samples immediately precipitates out easily as soon as chilled
     ethyl alcohol is added whereas, the others requires storage for about 15-20 minutes at -20C and also I wanted to
     know that what is the role of Ethyl alcohol in DNA precipitation. I will be very thankful to you if you help me out in


               shumaiza anis
               7 months ago
202. I am trying to precipitate DNA using the commonly used ETOH method.However, I can see that the salt has
     precipitated to the bottom forming a lot of white precipitates!Could you tell me if there is anything I can do.


               shumaiza anis
               7 months ago
204. i used ethanol precipitation. but at the lots of salt precipitated with DNA. is there any way to clean up the salt
     because the i have extracted the DNA from gastric biopsies.


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       6 months ago

               6 months ago
207. Hey there,
     would you tell me the soucre of your statement, that NaCl should be used when SDS is present in the preparation?
     Greetz, Chris (Karlsruhe Institut of Technology KIT, Germany)

                6 months ago
209. Hi Chris,
     Maniatis is one source and if you don’t have that (it’s old) then check the new Sambrook and Russel Molecular Cloning
     The appendix has a section on ethanol precipitation.

210. Suzanne
     (also- personal experience- I tried precipitating DNA that had SDS in the lysis with NaAcetate vs NaCl and without
     NaCl it was a mess.)


                6 months ago
212. THX a lot…I will check the literature. Ok, I’ll stay with my NaCl
     Greezt, Chris


                6 months ago
214. Hey

215. I have a question, do the PCR purification kits help in desalting DNA? Im doing something where Im eluting ssDNA with
     a buffer containing Urea…can i use these kits to get rid of the Urea? Or should i do a std ethanol precipitation? will
     that help get rid of the Urea? Im trying to avoid dialysis..


                5 months ago
217. Hi,
     I have extracted genomic DNA from my samples and quantified by using UV absorbance and fluorescence-picogreen to
     check the sensitivity of the quantitation method. The outcome was, DNA yield quantified by picogreen are almost 3
     times higher than the yield in absorbance. The ratios of A260/280 are within the range (1.6-2.2) but the ratios for
     A260/230 are very very low in range 0.28-0.5. I used salt out method to extract the DNA. Do you think the reason
     because it is high salt in my DNA or because I didn’t wait to dry the pellet after DNA precipitation with 100% EtOH,
     followed by 70% EtOH wash step before adding the TE buffer?
     Therefore, is it possible that I still have ETOH in my DNA which makes the DNA difficult to dissolve completely and
     only when quantified by picogreen which bind specifically with dsDNA it gives high yield?

218. From what I have read in previous paper, DNA yield by absorbance are higher compared to picogreen.

219. Please help me to understand this situation.

220. Thans a lot. Sorry for very long post…

221. moderator-edited post because of spelling errors.


                5 months ago
223. hello there… i have problem with my DNA precipitation. i’m isolating DNA from NPC cell line bdw. it really take me
     longer time to pellet the DNA after the addition of absolute ethanol. what might be the problems?? i need to do
     around 4 to 5 times of centrifugation at high speed for 10 mins to pellet the DNA. usually my dna will be sheared or
       degraded during this procedure


                Ali Nourizadeh
               5 months ago
225. hello ,Your explenation is so intresting
     many many thanks
     but what is the role of low tempreture in precipitation?


               4 months ago
227. Hi Nick

228. I have a qouestion. I am doing HRM analysis for SNP genotyping. Salt concentration of DNA samples has high effect in
     this analysis. the DNA pellets are dilouted in TE. Would you please help me how to precipitate salt from my Samples
     without extracting DNA again?
     Many thanks in advance


               4 months ago
230. Quick question – if the purpose of the salt is to neutralize the backbone charge, why is the final concentration of the
     different salts used so disparate?

231. Also, in trying to check calcs, if one uses 0.1X NaOAc (stock @ 5M)and 2-2X EtOH, I don’t calculate a final
     concentration of 0.3M…??? Example, resuspend sample in 1 ml, add 0.1 ml 5M NaOAc, add 2 – 2.5 ml EtOH. I get (0.1
     ml)(5M) = x (3.1 ml), x = 0.16 (or if using 2.5 ml EtOH, x = 0.14.

232. What am I missing?


               4 months ago
234. Sorry for being an idiot, I figured out the calcs moments after posting, it’s 0.1 volume of a 3M solution of 3M NaOAc,
     so the 0.3M “final” concentration is referring to the concentration of the salt in solution PRIOR to addition of EtOH.

235. Still curious about the different final salt concentrations though!


               Danilo J Bonsignore
               3 months ago
237. What is DNA is dissolved in carbonated water? Say, like Human remains in soda tanks in McDonalds. Would this
     method work? If the goal is just to show there are traces of Human remains in water, what would be the easiest way
     to make it evident?


               Sudam mane
               2 months ago
239. pls tel me that during preciptisation of RNA lithium chloride is added to prevent protien synthesis &DNA polymerase
     ,chloride ion plays key role here.tell me actual mechanism or actual role of chloride ion to prevent the protien
     synthesis & DNA polymerision


               Franco Liu
               2 months ago
241. I just want to ask, what effect does the ethanol extraction on the DNA extracted and the subsequent AGE band it

               2 months ago
243. I’ve been running 100 micro liter PCR reactions and getting plenty of 2007 bp product with none of the DNA held up
     at the origin when 5 microliters of it is run on gel. The problem comes when I go to extact the PCR product and get it
     dissolved up in water. Every time I try to dissolve my pellet back up into 100 microliters of water and run 5
     microliters on a gel there is a significant quantity of my DNA which refuses to enter the gel. It just stays hung up at
     the origin. Can you help? Below I detail my procedure.

244. I remove all of the PCR volume from under oil, bring it up to a 250 micro liter volume with water and then add 250
     micro liters of phenol saturated with 0.5M Tris-HCl (pH 8.0). The upper aqueous phase is clear but after it is
     transferred to a clean, new microcentifuge tube turns quite cloudy. I add twice the volume of 2% potassium acetate
     in 95% EtOH to the aqueous phase, vortex, and allow to precipitate overnight at -20 degrees C. I spin for 2 minutes on
     a desktop centrifuge and decant the salted Ethanol. The pellet is allowed to dry in a vacuum for 10 min (There is
     usually what appears to be a small droplet of something present in the tube with the pellet after drying). I then add
     100 microliters of water and allow the pellet to dissolve for 2 hours on ice with periodic vortexing every 15 minutes.
     Five 5 microliters is then removed and run on gel.
     Note: the small droplet alluded to above does not disappear even after 20 minutes of drying under vacuum and as yet
     I have not been doing any 70% washing of the pelleted DNA.


               Max Power
               2 months ago
246. Thanks a lot…


               1 month ago
248. Hi ! I am currently experiencing problems with RNA precipitation.

249. Here is what I do: I perform RNA extractions using TRIzol reagent with cells in suspension. I centrifuge them, discard
     the supernatant and then add 2 mL of TRIzol (for about 3-5 millions cells) and put the tubes at -80C O/N. The day
     after, I make new dilutions of TRIzol (about 500 µL of sample + 1.5 mL new trizol) to make sure all the cells are
     really lysed and I add 500 µL of chloroform for the extraction. After centrifugation 5min 12000g at 4C, I obtain the
     classical 3-phase separation and I transfer the aqueous phase in 2 new microtubes (300 µL/tube), then I add 900 µL of
     isopropanol to each tube.
     My problem is that after the centrifugation, I obtain a transparent gel-like pellet that does not stick on the side or
     bottom of the tube, but floats in the tube.

250. What is wrong? DNA contamination due to too much chloroform ? New dilution of trizol is unnecessary? too much
     isopropanol??? should I use ethanol instead?

251. thanks


               1 month ago
253. Hello Marie!
     I think you’re overkilling with isopropanol. According to the Trizol protocol, you should add 500ul of isopropanol for
     each 1ml of Trizol used. This is based on the assumption that you’re going to retrieve 450-500ul of RNA-containing
     supernatant for each 1ml of Trizol.
     Since you’re saving only 600ul of supernatant (why so few?), and you split it into two 300-ul aliquots, you should add
     300 ul of isopropanol to each tube.
     Also, you should follow the protocol, there’s usually no need to tweak it at all. I.e. you’re adding 500ul of
     isopropanol (while 400 ul is the correct amount for 2ml Trizol, which is roughly what you have after the dilution).
     Finally, it’s really best to make sure that the cells are thoroughly lysed in the first day. If cells lyse incompletely,
     their RNA can meet its end against an undenatured RNAse before escaping into the Trizol solution. So if you’re going
     to add more Trizol anyways, it’s just best to add it straight away when you’re lysing the cells.
     Best luck!


               1 month ago
255. Thank you Sir, I have precipitated DNA many times but I did not know the exact function of ethanol and salt. So
     thank you again for your deep explanation.


              27 days ago
257. Hi,
     I have a DNA extraction protocol that tells you to add Glycogen to the isopropanol prior to adding my lysed and
     protein precipitated supernatant, to enhance gDNA yield for compromised blood samples.
     What I’d like to know is whether you can add the Glycogen to the isopropanol after having already mixed the the
     supernatant with the isopropanol, and still get the desired increased yield?
     The reason for this is that I do not want to go to the expense of adding molecular grade glycogen to every sample,
     just those where I cannot see any stringy clump of DNA precipitated out from my supernatant by isopropanol.
     Many thanks


               26 days ago
259. Trying doesn’t hurt. In your case though, I don’t know if it’s the right thing to do. The background idea is that
     glycogen will start nucleation (i.e. formation of many tiny precipitate particles) and then the DNA will stick there,
     helping to grow the particle and precipitating along. So if the glycogen isn’t well dispersed, it’ll form few nuclei, and
     the increase in yield won’t be as much. You’d need to make sure that the glycogen forms very small precipitate
     particles, say, by vortexing very well – but if you’re extracting gDNA… that’s against your interests, I guess?

260. If I might suggest an alternative, linear acrylamide works as well as glycogen and is cheap, quick and trivial to make.
     I use it with good results. I follow this protocol:


               Kenneth Mitton
               25 days ago
262. We have added glycogen after the isopropanol (1 vol) (or 2 vol of ETOH), and it will increase your yield. What small
     amount of DNA you have that will resist precipitation will have to share that soluble compartment with the glycogen.
     You can add it at 37 C to your samples, mix, and then cool them down again.

263. You also benefit from the post precip wash steps because the larger more intact pellet you get will prevent those
     thinner DNA residues from fragmenting and becoming physically washed away during the 70% ETOH washing.

264. People think the yield is all about the precipitation step, but I am constantly showing staff and students how I can
     recover twice the DNA they recover from the same solutions simply by taking much more care of the pellet (or
     residue) once you get it. I make my staff “soak” (wash) three times, not twice, and not try to take off every uL of
     wash. The last bit dries away fast in the spin vac.


               22 days ago
266. Hey! Great article, great discussion!

267. Simple Question: i run a genemole RNA-Extraction and i got my totalRNA sample in TRIS (100µl). I need it desalted for
     RNA-Chip analysis but be especially interested in microRNAs, so i fear any loss!


               22 days ago
269. What kind of desalting or precipitation do you suggest?


               20 days ago
271. concerning LiCl precipitation, Ambion has a nice webpage:

272. it looks like it doesn’t really inhibit translation (also, you apparently don’t need to add ethanol, just 2.5M LiCl is


              16 days ago
274. Hi Nick,
     great article.
     Can you make up sodium acetate and store at 4C or -20C for long periods??


               16 days ago
276. Hi Natasha,

277. Yes, I normally make up a solution and store small aliquots at -20

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