Hello.....I have used glycogen for RNA isolation (Trizol protocol) in purpose of next generation sequencing. It worked perfectly though it is difficult to see the small RNA pellet. Then, I tried to use glycoblue for better viewing of the pellet and its easier to see the pellet. But I could not get any band after cDNA synthesis from that isolated mRNA. Does anybody have idea why glycoblue does not work perfectly?
Thanks
I have used the Takara reagents (Trizol protocol) and found the perfect pellets work for the Illumina sequencing. I suggest you to use the reagents and follow some precautions and check the quality by the Nano drop. Hope you can handle the problems.
I have used both glycogen and glycoblue, and have found no adverse effect on cDNA synthesis from either. Perhaps check to make sure that the reagent that you purchased is guaranteed RNase-free. If it is, then look to your other reagents as the cause for your cDNA synthesis failure - could anything else be contaminated? Is your RNA polymerase still fresh, etc? I would check these things before worrying too much about the glycoblue. Good luck!
Do also note that with glycogen or glycoblue, a pellet is not a guarantee of RNA.
Thanks for your answer. Could you please write me how much or what concentration of Glycoblue have you used for RNA isolation?
RNA polymerase is fine and we also use RNAase inhibotor during cDNA synthesis. So it should not be a problem.
Halo Sumanta,
There is no difference between the two glycogenes. Only when you run it on a gel or measure it on a nanodrop it could maybe make a very small difference in the color of your band or 230nm values respectively. But this is neglectable.
Most people add 1 or 2 uL. The rule of thumb is that you should never let the amount of uL be more than 1/10th of your final RNA solution volume. (e.g. if you elute your RNA in the end in 10uL the maximum of Glycoblue you can use is 1uL)
Thus, the missing band has nothing to do with your glycoblue.
Good luck!
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