Good video and explanation! FYI for those in comments, it's good to air-dry RNA by leaving the tube on its side (on a kimwipe), as this prevents contaminants from settling in the tube. Also good practice to make aliquots of your dH2O that you draw from, so you can limit the number of times you remove water directly from your DEPC water stock.
Many thanks for this informative video. But I have a question about the precipitation of isopropyl alcohol. I read from other papers that the function for isopropyl alcohol is to lower the dielectric constant so that RNA could precipitate. But not for neutralizing the ions of RNA. Could anyone help me with this question?
I thought the organic phase layer would be composed of mostly chloroform rather than phenol, because Chloroform is a much more dense and heavier compound than phenol. ??
Hi...i m working on gene expression for that i hv to isolate plant RNA for cDNA preprtn...can u share protocol of this method...n plz tell me shud i go for this method or RNA extraction kit for plant RNA isolation...
Very informative, but i have a question. After we put phenol chloroform and there's aqueous layer and organic layer, do we discard the organic layer first? And then add the isopropyl alcohol?
+nur hafizah The aqueous layer is the clear layer on top, the organic layer is the reddish layer on the bottom. Your RNA should be in the top aqueous layer and is simply transferred by pipetting into a new tube. The remaining organic layer in the old tube contains your unwanted cellular debris and DNA and can be discarded. The ethanol precipitation step of your desired RNA is then performed on the aqueous layer that you had transferred into the new tube.
Hello,I did not understand what actually is the use of phenol in TRISOL.Its use is only to be an organic dissolver for the other polar molecules?Our professor in laboratory used to say this is to "hold" the debris and DNA.Also ,why and when do we put chloroform in the whole process?Also, EtOH has the same use of isopropanol?
Your professor is right by saying phenol-chloroform mixture holds down protein and hydrophobic lipids in the denser organic phase. . You add the chloroform (my lab uses BCP as it is less dangerous) straight after homogenising cells/tissue in trizol to separate the phases. Isoproponal is used to precipitate DNA/RNA as they are insoluble. To do this with EtOH you would need to add sodium acetate to perform a precipitation. Other times we use EtOH is to clean the pellet, not precipitate.
I believe the heating to 55-60C step you are referring to is simply to assist in resuspension of the RNA pellet because depending on the amount of RNA you have in your pellet, there may be difficulty in dissolving the RNA into solution in water.
I believe the heating to 55-60C step you are referring to is simply to assist in resuspension of the RNA pellet because depending on the amount of RNA you have in your pellet, there may be difficulty in dissolving the RNA into solution in water.
Sorry for the delayed reply. The TRIzol reagent should already be maintained at a low acidic pH (~4), thus you should not have to worry about the pH during the extraction process. If you are encountering issues with the pH of your TRIzol reagent, you may have to either get a new bottle or make your own "TRI" guanidium-acid-phenol reagent (protocol here: www.openwetware.org/wiki/RNA_extraction_using_self-made_guanidinium-acid-phenol_reagents). The low acidic pH of the TRIzol reagent (~4) is essential, or else you will get both DNA and RNA in your aqueous phase.
See the first results item after you google "RNA extraction centrifuge speed". Or look at item 11 from www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwjiuI2Q9__XAhXIkOAKHb6DB1UQFggrMAE&url=https%3A%2F%2Fwww.scripps.edu%2Fcalifornia%2Fresearch%2Fdna-array%2Fpdfs%2FRNA%2520Extraction%2520Protocol.pdf&usg=AOvVaw3YtdKDVTLN4PRPlhDTXmAV
A more accurate equation is g = (1.118 × 10^-5)*R* S^2, where R = radius of the spinning rotor and S = centrifuge speed measured in revolutions per minute (RPM). g is the local gravity this spinning produces. SO when a centrifuge is spec'd at 12,000g, it's an accurate representation of the amount of acceleration the RNA solution is experiencing.
This is the most perfect thing I have ever watched
Biochem is fun. Micro is funner.
Good video and explanation!
FYI for those in comments, it's good to air-dry RNA by leaving the tube on its side (on a kimwipe), as this prevents contaminants from settling in the tube. Also good practice to make aliquots of your dH2O that you draw from, so you can limit the number of times you remove water directly from your DEPC water stock.
I am so thankful this video exists! You explained it really well!
Felt like I was a kid again learning through cute drawings and sketches ❤ really awesome 💖
This is awesome. i hope it works well for plant samples as well.
One of the best tutorials a have seen so far! (and I saw a lot) Great job :)
Cheers from the LMU Munich, I'll check the other videos ;)
you are very professional.hope we can discuss further,my email:zgzhou@hebeismart.com
the cutest rnase that ever liveddddd
This is such a great video. I love the animations and it is explained very well. Thank you.
Thank u so much for such a peaceful and informative video
I just cleared all steps now by ur video..keep it up
This is wonderful. Thank you so much!
Clear and precise explanation! Thank you
Omg I understood every concept quite clearly thank you for making it it's so helpful 💜😍
Great video! I have a presentation on RNA isolation and this helped a lot!
Awesome video! I appreciate these types videos so much
Thank you so very very very much for this amazing and wonderful explanation 👍😎
Nice video!!
Can you please explain how pH less than 4 will allow only RNA to be in the aqueous layer and not DNA.
thanks
Absolutely informative! Thank you so so much!
It is one of the best clearly explained with funny animation videos I have ever seen
Thanks this video is very informative!
Thanks a lot for this video! It was helpful.
Thanks, that was very well explained.
Many thanks for this informative video. But I have a question about the precipitation of isopropyl alcohol. I read from other papers that the function for isopropyl alcohol is to lower the dielectric constant so that RNA could precipitate. But not for neutralizing the ions of RNA. Could anyone help me with this question?
Very well explained, Much better than my tutor
Great！RNA Extraction may use guanidine thiocyanate is used for RNA isolation and can solubilize proteins
Thanks a lot for this wonderful tutorial
Thanks a lot! It was really helpful
love the cartoon visuals !
Thanks for this great theoretical ans practical explanation.
It's really informative. Thank you
AMAZING. THANK YOU SO MUCH.
Thank you for the very useful video.
Nicely done and explained.
Excellent! I will use it with my students.
Thank you for your help 🙏
A very informative video tutorial.
Many thanks for this informative video.please upload more informative video like this.
very useful, thank you.
Appreciate well-explained technique
I'm a chemist and this made sense so good job and thanks!
Will it work with viral RNA? Would be the same process?
The same or very similar?
How do you prepare bacteria sample for RNA extraction if you have plated culture?
Yea... Very much informative. Thankyou
Nicely done ! Very helpful . Can you do more such lab based experimental videos ?
Thank you!
Can you please state the reason, why RNA is isolated at pH 4 and DNA at pH 5/6?
Thanks! Very useful!
awesome video. thanks!
nice video very informative
you don't need to put the samples on ice and work on ice all the time?
I thought the organic phase layer would be composed of mostly chloroform rather than phenol, because Chloroform is a much more dense and heavier compound than phenol. ??
Hi...i m working on gene expression for that i hv to isolate plant RNA for cDNA preprtn...can u share protocol of this method...n plz tell me shud i go for this method or RNA extraction kit for plant RNA isolation...
!! it's very helpful!!
it's a great tutorial thanks a lot
Among the favorite ones 👍👍👍
Very good explanation.
I wish our trizol was red! Ours is yellow so it is much harder to see the difference in phase layers :(
Very informative....thank you
Thanks so much.
which sample is more suitable rather than bacterial cell culture..please reply
thanks ! it was useful!
Very informative, but i have a question. After we put phenol chloroform and there's aqueous layer and organic layer, do we discard the organic layer first? And then add the isopropyl alcohol?
+nur hafizah The aqueous layer is the clear layer on top, the organic layer is the reddish layer on the bottom. Your RNA should be in the top aqueous layer and is simply transferred by pipetting into a new tube. The remaining organic layer in the old tube contains your unwanted cellular debris and DNA and can be discarded. The ethanol precipitation step of your desired RNA is then performed on the aqueous layer that you had transferred into the new tube.
Hello,I did not understand what actually is the use of phenol in TRISOL.Its use is only to be an organic dissolver for the other polar molecules?Our professor in laboratory used to say this is to "hold" the debris and DNA.Also ,why and when do we put chloroform in the whole process?Also, EtOH has the same use of isopropanol?
Your professor is right by saying phenol-chloroform mixture holds down protein and hydrophobic lipids in the denser organic phase. . You add the chloroform (my lab uses BCP as it is less dangerous) straight after homogenising cells/tissue in trizol to separate the phases. Isoproponal is used to precipitate DNA/RNA as they are insoluble. To do this with EtOH you would need to add sodium acetate to perform a precipitation. Other times we use EtOH is to clean the pellet, not precipitate.
thank you
Can you please give the reference for the described method?
Exactly it's much more informative
te amo, me salvaste el pellejo muak!
Thankyou for the video..
thanks a lot, wery helpfull
Where are the captions?
I have no idea what this is but it's very interesting.
Doesn't to be on ice?
Sir, can you please tell how we can take O.D.( optical density of RNA) of isolated RNA ?
hope we can discuss further ,my email:Zgzhou@hebeismart.com
thanks!
incredible
great! I have also seen that after the resuspension in DEPC water there is a 10-15 minute incubation at 55-60 degrees. Is that really necessary?
I believe the heating to 55-60C step you are referring to is simply to assist in resuspension of the RNA pellet because depending on the amount of RNA you have in your pellet, there may be difficulty in dissolving the RNA into solution in water.
I believe the heating to 55-60C step you are referring to is simply to assist in resuspension of the RNA pellet because depending on the amount of RNA you have in your pellet, there may be difficulty in dissolving the RNA into solution in water.
I see, thanks for the swift reply!
ChangBenson
very good！
so good n thanks
Thank you so match
please mention amount of ip, trizol and other ......
Can i freeze cell in -20 Celcius for 8 hours, then into liquid nitrogen for 3 months before i do the RNA extraction? anyone can help me on this?
No. You have to immediately put the samples in liquid nitrogen
how do we maintain pH 4 in this extraction?
Sorry for the delayed reply. The TRIzol reagent should already be maintained at a low acidic pH (~4), thus you should not have to worry about the pH during the extraction process. If you are encountering issues with the pH of your TRIzol reagent, you may have to either get a new bottle or make your own "TRI" guanidium-acid-phenol reagent (protocol here: www.openwetware.org/wiki/RNA_extraction_using_self-made_guanidinium-acid-phenol_reagents).
The low acidic pH of the TRIzol reagent (~4) is essential, or else you will get both DNA and RNA in your aqueous phase.
thank you for this video
Bk to दर हूं धनं धन
how to make equipment RNase free
masonic pyramid
very informative. Thanks
un traductor al español porfa
wow!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!111
it 12g, also showed 12 rcf on the centrifuge. NOT 12000G！
Wrong! It's 12,000g in his case. The centrifuges in our labs spin at 11,000g.
David Musoke wow that's so amazing. Ur centrifuge can create a black hole.
See the first results item after you google "RNA extraction centrifuge speed". Or look at item 11 from
www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahUKEwjiuI2Q9__XAhXIkOAKHb6DB1UQFggrMAE&url=https%3A%2F%2Fwww.scripps.edu%2Fcalifornia%2Fresearch%2Fdna-array%2Fpdfs%2FRNA%2520Extraction%2520Protocol.pdf&usg=AOvVaw3YtdKDVTLN4PRPlhDTXmAV
g=(1.18*10^-5)RPM.
A more accurate equation is g = (1.118 × 10^-5)*R* S^2, where R = radius of the spinning rotor and S = centrifuge speed measured in revolutions per minute (RPM). g is the local gravity this spinning produces. SO when a centrifuge is spec'd at 12,000g, it's an accurate representation of the amount of acceleration the RNA solution is experiencing.
A very informative video tutorial.