I run E coli total RNA on 10% TBE-Urea PAGE denaturing gel, and just wanted to know what RNA species these bands exactly are.
I used an RNA ladder contains ssRNA of 1000, 800, 600, 400, 300, 200 and 100 nt, respectively. The 1st, 2nd and 9th, 10th lanes are E coli total RNA from source #1; lanes 4th, 5th, 6th and 7th are E coli total RNA from source #2. Both are commercial products since I didn't have the things ready to culture E coli in the lab.
I am focusing on 5S rRNA and tRNAs. There are three bands around the 100nt ladder, one of which almost matches with the 100nt ladder, the other two are slightly longer. I supposed one of the longer two is 5S rRNA (~120 nt), but what are the other two?
I suppose the three bands smaller than 100nt are tRNAs, because E coli has tRNAs of 70~90nt, but it would be helpful if someone could tell me exactly what these tRNA species could be.
Also, it seems that these three
There is no way to know exactly which tRNA molecules are on that gel unless you gel-purified and sequenced them. Does it matter for your project?
Also, those ~100 nucleotide bands are all the same size in all of the wells. Use the ladder as a guide and you'll see that any minor difference in distance from the well is explained by small variations between wells, not an overall difference in product sizes.
Look, it's a better use of your time to show this gel to someone in your lab. We don't know your overall project, but I'm guessing that figuring out identities of the bands on this gel is not it. What question are you asking?
Katie A S Burnette Thanks for your answer! I do, however, wanted to figure out the identity of these bands, and I didn't get good answers from our lab members, so I am asking people in here for help.
What I am trying to do is to isolate these individual RNA species and our lab is developing a method to de novo sequence them using a mass spectrometry-based method. With that said, I wanted to identify these RNA bands so that I can choose which one to recover and have an idea what is being sequenced.
Best,
Weichen
Well, you could try doing some blots with specific probes to some of your top candidates. If what you need are decent quantities of know RNAs then you could just produce specific ones rather than using a bulk sample such as this one. Use an RT kit to make up larger quantities of known RNAs and use those to get your new protocol developed and optimized.
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