I am doing an rna extraction from tomato leafs, using the Trizol protocol. But when I add chloroform, and centrifuged the samples, the hidrofilic portion gets a pink coloring. And the color keeps going in the steps. Any one know what it is? I am afraid that could be Guanidinium thiocyanate, and could be resulting in a low proportion of 260/230. Any information is valid.
Some cell components might have picked up red color from TRIZOL Reagent. See the attached document (yellow highlights). They offer a way to avoid it. You can try and find out.
I did this before, because my sample is full of cellular debris. Even then, was still pink . =p But thanks anyway. nice document
Hi,
did you grind the tomato leafs in liquid nitrogen?In this case add 10 ml TRIZOL reagent to one gram sample. Also you can use TRIzol method followed by lithium chloride (LiCl) precipitation and DNase treatment.
I used 50-60 mg of tomato leafs, and 900ul of Trizol. What is the purpose of using lithium chloride? Thanks
People are suggesting using chloroform for treatment. See the link for discussion ("Removing phenol contamination from total RNA"): They also had a pink tinge at the "clear aqueous layer". You can try and find out.
http://www.protocol-online.org/biology-forums-2/posts/7291.html
LiCl increase yield and stability of the RNA preparation for tissue samples especially plant species and improve cDNA synthesis.
You can add more than once extraction step with chloroform too.
How old is the chloroform that you have? How long are you centrifuging the sample for following chloroform addition?How much did you add chloroform?all are the key factors that influence chloroform extraction step.
following papers may help you
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- Science method