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Denaturing agarose gel electrophoresis

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Denaturing agarose gel electrophoresis

Postby Katy_Bobbles » Fri Sep 21, 2007 7:30 pm

Hi all

When I did denaturing AGE of purified RNA samples, 28S and 18S ribosomal subunits were included in the gel as markers.

I understand that rRNA is the "first" form of RNA in central dogma but how does that relate to my RNA? Why doesn't it convert to tRNA then mRNA.

Sorry if what I've just said sounds stupid but I'm a newbie to all this! I've been very confused this week as we have had to cram in lots of different methods in 4 days. Google is not as good in honours year because the stuff is so advanced!

Any help would be appreciated! :D
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Postby blcr11 » Fri Sep 21, 2007 8:04 pm

The Central Dogma is “DNA makes RNA makes Protein.” But I think you may be confusing the intentions of the dogma. DNA encodes the gene sequence; the gene sequence of the DNA is transcribed into specific messages or mRNAs that find their way to the ribosomes where the messages are translated into protein. I’m not sure why you think “your RNA” would convert to tRNA. I’m not sure what you ran on your gel besides the markers, but if you had mRNAs, they will form a size-related smear along the track with shorter molecules running faster than longer ones. You denature the samples so they will not have any secondary structure that might alter the electophoretic mobility—sort of like why you do SDS-PAGE of proteins to see things run out according to their molecular weight and more-or-less independent of charge or shape. Usually you transfer the gel to a blot and probe the blot with a labeled oligo that recognizes a specific mRNA. That message should light up as a band of the correct size if the message is present and you’ve run the gel correctly. But I don’t know if this is the experiment you’ve actually done.

When Crick first stated the dogma, tRNAs had not been discovered and ribosomal RNAs are not themselves translatable into protein; rather, they are the catalytic surface provided for the translation machinery which includes mRNA, acyl-tRNAs and a host of additional protein factors.
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Postby Katy_Bobbles » Sun Sep 23, 2007 10:02 am

I'm understanding better now Bcr11.

I think I was talking out of my bum when I was talking about the tRNA. :oops:

Thanks again :)
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Postby Katy_Bobbles » Sun Sep 23, 2007 4:37 pm

Sorry same topic different question.

OK it turns out there were no markers added to the gel. We were not supposed to which was weird.
Instead the gel was photographed with a fluorescent ruler. I don't get how we are supposed to do it with no markers. I mean it is easy to tell that the 2 bands I have are the 28S and 18S bands but how do I explain it in a report?

What have I to compare it to with no markers. I have the size and amount of DNA hyperladder legend but that is obviously not the same size as the gel. :?
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Postby blcr11 » Mon Sep 24, 2007 1:40 pm

http://www.ambion.com/techlib/append/supp/rna_gel.html

So basically you did the above. I’m not sure what the purpose of your experiment was supposed to be, but in the link above it was simply to show the difference between good quality RNA preps (those with clear bands for 18S and 28S rRNA) and poor quality preps (no such bands and everything is smeared out with a number average size at or near the front of the chromatogram.) This “flourescent ruler” you refer to couldn’t possibly be RiboRuler™ could it? This is just a MW ladder for RNA similar to DNA markers available for DNA gels. I guess you should be able to say at least two things about your 18S and 28S bands. Namely, that, if you see clean bands you can be assured that the RNA prep is intact and not significantly degraded. And, assuming the ladder is a size marker like RiboRuler™, you should be able to say roughly how many bases there are in each of the two bands. Beyond that, I don’t know what else there is to say about the experiment. The size of the bands for the High Range ruler are 200, 500, 1000, 1500, 2000, 3000, 4000, and 6000 bases. Different vendors will supply different sized markers, so you'll have to check your source for the actual sizes on your gel (if, in fact, that's what you have--I can't imagine a literal ruler marked off in mm or cm and covered with fluorescent material).
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Postby Katy_Bobbles » Mon Sep 24, 2007 2:20 pm

Thanks for your help bcr11. Much appreciated! :D

Yeah I saw that page. It was the only one I think that kind of explained it properly but they had markers and I don't thats how it is so hard.

Thats good then because I have 2 distinct band it suggests my RNA is intact. Yay I got 1 thing right.

Its definately not RiboRuler™. Wish it was because it would be easy to do.

And better start imagining because it is a fluorescent ruler! It's like a normal ruler but the cm and mm are written in some sort of fluorescent ink or something! :)

But I don't know if I'm supposed to work out the number of bp per mm on the gel using the fluorescent ruler and the number of bp per mm on the legend. Seems a higglety pigglety way to do it but it may be the only way! Why couldn't we have had Riboruler or some other markers! :x
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Postby blcr11 » Mon Sep 24, 2007 3:07 pm

I guess you can always report Rf values for the bands. Rf is defined to be the ratio of the distance the band moves to the distance the front moves over the same amount of time. This number is a hold-over from the old days of paper chromatography and was used to characterize the mobility of species on the chromatograms. Things that move quickly will have Rfs approaching 1 while things that move slowly will have Rfs closer to zero. The Rfs for the two bands should at least be different and, I'd guess maybe somewhere in the range of 0.3 to 0.6--but that is just a really wild guess on my part. Without seeing the actual distances I can only guess. I supppose you can always just say that the 18S band was at so-so cm while the 28S band was at so-so-sum cm. Not very enlightening, but at least it describes the observation.
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Postby blcr11 » Mon Sep 24, 2007 3:39 pm

If you know the relationship between mm or cm of movement to number of bases, you can certainly estimate the number of bases in the rRNA bands. I'm a little surprised that they had you put a DNA ladder on an RNA gel--or do I misuderstand. You mentioned something about the legend giving you a relationship between mobility and the number of bases (it should be bases, not base pairs; you ran a denaturing gel; there should not be any base-paired nucleotides present except for maybe some snapback hybridization as you stained the gel.)
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Postby Katy_Bobbles » Mon Sep 24, 2007 7:40 pm

There was no ladder on the gel. From what I could see we have the DNA hyperladder as the legend. So maybe we are not supposed to use that. I don't know. There was a lot going on in the lab and that particular part was not made very clear by the lecturers.

Maybe we have just to say that the RNA migrated with the 28S and 18S bands? We have to mention if distinct or less distinct bands. How many.

Well I have 2 separated distinct bands. That means the RNA is intact. The RNA has been separated into the 28S and 18S subunits. THE END! Maybe thats it! :lol: Doubt it though!

I thank you again bcr11 8)
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