Hey, I am isolating RNA from the IVT product. Earlier I used to extract by adding simply chloroform and precipitation by Isopropanol. However, the yield comes in ug but in gel, the RNA is 1/10 of the nanodrop reading. Sometimes, there is no RNA on gel.
Then i followed Trizol method (Followed by Senior) , Here I add the Trizol in 100ul of IVT sample and adding chloroform. However, there seems the Trizol layer on the top which doesn't go away by adding more Water or Trizol? What should I do? I need a good RNA for my experiments.
Are you using DNase to remove the DNA template after transcription? If not, you may be seeing the template on the Nanodrop. Another possibility is that unincorporated nucleotides are making the Nanodrop reading higher than you see on the gel. Additionally, when you do the Nanodrop, are you blanking with the same buffer your sample is in?
When I'm purifying RNA from IVT, I typically run it on a denaturing PAGE gel, cut the bands, then elute the RNA from the gel pieces. For larger RNAs, I do LiCl precipitation. You can also look into commercial spin column-based kits.
I give DNase treatment and then I purify RNA by adding chloroform and isopropanol. I assume the same that incorporated nucleotide giving high reading. However I use same process to purify RNA from IVT for my different experiments which gives the correct nanodrop reading and bands on gel. However here I first did IVT and then Poly A tailing.. and then i am having trouble isolating good RNA.
Jyoti...
RNA isolation from IVT products:
Ensure RNA integrity with a Nanodrop or Bioanalyzer.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology