Reading the protocol - the volume of Trizol needs to be titrated according to the volume of cells to be obtained, and whether the cells have been grown in monolayer or suspension. Trizol is quite powerful so can degrade the cells and RNA altogether. Don't use 1 mL Trizol uniformly - only the volume you need. The volume of Trizol affects the volume of Chloroform, Isopropanol and Ethanol to be added later in the reaction.
I am using Trizol method for total RNA isolation from plant leaf. From my result, the nano drop reads 260/230 and 260/280 is equal 2.163 and 2.00 respectively. The quality on gel was shown no distinct bands! The RNA is degraded. Actually my sample is not fresh sample and was stored on deep(-80 degree C)freeze more than six months. what is the causes of RNA degradation? Is degraded RNA can be used for cDNA synthesis for qRT-PCR analysis?
When you say 3 layers if you find one organic layer(pink), an aqueous layer(RNA) and fibers in between those two layers then it's correct the fibrous layer is DNA
Hello, I have two questions 1. After collecting my sample and adding Trizol, can i store it in -20C for a month? 2. Why we must change centrifugation 12000g to 7500g in etoh wash step? Can i perform this step with 12000g?
I am using 200 mg of sample for 2 ml trizol I get thick pellet but it gets dislodge after ethanol wash and sometimes decant with the ethanol please suggest what should I do that it doesn't get dislodge from the bottom?
for most applications you shouldn't need DNAse with this method. The RNA and DNA are separated in distinct layers after the chloroform centrifugation so the amount of DNA in the aqueous phase should be low. If you really needed to make sure you have no DNA I guess you would treat the final product with DNAse.
@@noor14-5 Late here, but in case someone reads this. It shouldn't affect the concentration per se, but totally dry RNA is VERY difficult to completely dissolve in water, and having undissolved RNA will affect the concentration. Completely dry RNA has to be dissolved in a shaker at 37C, and you may still have to further use pipetting a few hundred times after that. All that could worsen the quality of your RNA so it's much better to simply not let it dry that much. Just remove as much ethanol as you can without disturbing the pellet and let it air dry for 5-10 minutes.
@0:09 to the sample... which sample????? are all samples treated the same way as in here??? @2:02 wash pellet... where is the pellet??? @1:56 what is happening exactly here?? emptying all the content??? why why the music!!! Speak instead, give instructions !!!
1.General protocol for all samples, can be modified according to what your sample is. Wash pallet- in some cases pallet is not visible, but it's there. I wasted lot of samples thinking there is no visible pallet. 3.Not emptying content. It is referred to as washing the pallet. We wash it with isopropanol Or ethanol and then remove them.
After centrifugation your precipitated RNA will be at the bottom of the tube as a white pellet. As the other person said, even if you don't see it you could have some RNA. At 1:56 the supernatant is discarded and the RNA pellet sticks to the bottom of the tube.
Great video, it is very useful, we are procuer of guanidine thiocyante, we can give some advise to our customer after learn this very ,thank you very much.
Reading the protocol - the volume of Trizol needs to be titrated according to the volume of cells to be obtained, and whether the cells have been grown in monolayer or suspension.
Trizol is quite powerful so can degrade the cells and RNA altogether. Don't use 1 mL Trizol uniformly - only the volume you need.
The volume of Trizol affects the volume of Chloroform, Isopropanol and Ethanol to be added later in the reaction.
Why the upper phase is red, phenol-chloroform
phase and the lower phase is aqueous ?
This technique is really great !!!!!!! Loved the video :)
I am using Trizol method for total RNA isolation from plant leaf. From my result, the nano drop reads 260/230 and 260/280 is equal 2.163 and 2.00 respectively. The quality on gel was shown no distinct bands! The RNA is degraded. Actually my sample is not fresh sample and was stored on deep(-80 degree C)freeze more than six months. what is the causes of RNA degradation? Is degraded RNA can be used for cDNA synthesis for qRT-PCR analysis?
RNAs are highly unstable they get degraded very quickly even at -80•© .... Better to prepare cdna and then store it for further processing.
I need your help
I hope that you're using a hood when using chemicals like trizol and chloroform! :O
Exactly
@@noor14-5 fv
One question why the sample with trizol and chloroform was incubated on ice not room temperature? I always do on room temperature
To avoid degradation by RNAses.
After adding isopropanol and centrifuging it, could you see a white clear pellet? Please help
Can you please help me with protocol for trizol method from whole blood
The best if you make the reverse transcription,then store the sample,because the RNA is really sensitive.
When I do the same procedure, I obtain 3 phases after the chloroform and triazol, is it correct? It is a sample from adipose tissue.
When you say 3 layers if you find one organic layer(pink), an aqueous layer(RNA) and fibers in between those two layers then it's correct the fibrous layer is DNA
"Hey can you tell me if this rag smells like chloroform?"
After centrifugation with isopropanol, I get 2 layers? Is it ok
@BrianK0220 you get an alcohol reaction which may damage the RNA
Can I use the same protocol for fungal spores without liquid N2?
Hello, I have two questions
1. After collecting my sample and adding Trizol, can i store it in -20C for a month?
2. Why we must change centrifugation 12000g to 7500g in etoh wash step? Can i perform this step with 12000g?
can i use this precedure for total RNA extraction from brain tissue
can i use the same protocol for plant material
Why we incubate with ice?? To prevent degradation Of RNA??
yes
This extraction can be done from a whole blood sample in EDTA.
Rafael Azevedo what will be the ratio of whole blood and trizol
Trizol needs to be 3 times of any sample. So for 10microlitre whole blood, you'll need 30microlitre trizol
what are theRNA extraction (TRIzol method) from the cells disadvantages
song name?
is it applicable for cell lines RNA extraction?
Just a short question.. after the extraction can I put the RNA in the -4°C and use it when I need it? Or it will compromise the RNA? Thanks!!
-80 is recommended
I am using 200 mg of sample for 2 ml trizol I get thick pellet but it gets dislodge after ethanol wash and sometimes decant with the ethanol please suggest what should I do that it doesn't get dislodge from the bottom?
Use a pipette to remove the supernatant. DON'T DECANT!!!
You need at least -80 degree fridge for RNA medium-long term storage.
the dnase treatment should be before or after the phenol chloroform
for most applications you shouldn't need DNAse with this method. The RNA and DNA are separated in distinct layers after the chloroform centrifugation so the amount of DNA in the aqueous phase should be low. If you really needed to make sure you have no DNA I guess you would treat the final product with DNAse.
If I air dry RNA until the pellet become transparent, is it going to affect the concentration of the RNA or the result?
Same question
@@noor14-5 Late here, but in case someone reads this. It shouldn't affect the concentration per se, but totally dry RNA is VERY difficult to completely dissolve in water, and having undissolved RNA will affect the concentration. Completely dry RNA has to be dissolved in a shaker at 37C, and you may still have to further use pipetting a few hundred times after that. All that could worsen the quality of your RNA so it's much better to simply not let it dry that much. Just remove as much ethanol as you can without disturbing the pellet and let it air dry for 5-10 minutes.
Thank you, this is really a very helpful guide to the protocol that comes along with the TriZol reagent.
thanks
Why can't they at least do the chloroform under the chemical hood? Most of these videos never mention it. Safety please!
By these method IAM getting 3 bands In gel but OD is coming 1.3-1.5 what is the problem......?
I guess you mean the ratios are those values, which would probably be phenol contamination from the trizol.
@0:09 to the sample... which sample????? are all samples treated the same way as in here??? @2:02 wash pellet... where is the pellet??? @1:56 what is happening exactly here?? emptying all the content??? why why the music!!! Speak instead, give instructions !!!
1.General protocol for all samples, can be modified according to what your sample is.
Wash pallet- in some cases pallet is not visible, but it's there. I wasted lot of samples thinking there is no visible pallet.
3.Not emptying content. It is referred to as washing the pallet. We wash it with isopropanol Or ethanol and then remove them.
After centrifugation your precipitated RNA will be at the bottom of the tube as a white pellet. As the other person said, even if you don't see it you could have some RNA. At 1:56 the supernatant is discarded and the RNA pellet sticks to the bottom of the tube.
I’m not seeing white pellet after centrifugation of isopropanol, does it means that I still have some RNA at the bottom ? Please I need help ?
was the sample treated with liquid N2?
I did it on liquid nitrogen but it doesn't matter RNA concentration and purity is same even without
Thank you so much I did the same as demo...
Thanks so much.
I will work in the lab with this music every day
Thank you for your help!! :D Happy Easter
Great video, it is very useful, we are procuer of guanidine thiocyante, we can give some advise to our customer after learn this very ,thank you very much.
thanks a lot this is really a helpful video
Kubota centrifuge. Like the most famous flip flops. Very nice music.
gud one.............
Hello and thank you for this practical clip. I have difficulty extracting spinal cord RNA and I can not get an accurate OD. Can you help?
Thank y very much
thank you,it was really useful.
gg
music is obnoxious.
Thanks a lot!!! :D
Good tune lol
Why why why the fucking disturbing music!!! why!!!
you need to have RNase Inhibitor in the end. 1 ul for every 30 ul of water.
that's what the trizol is for
Nuclease free water?