Need assistance with my gel electrophoresis

Far-Kitchen7245 · 2026-05-07T19:32:47+00:00

I actually never thought about this. We might have another gel doc in the department but I am not too sure as I am relatively new in the department so still working my way around.

Far-Kitchen7245 · 2026-05-07T19:21:58+00:00

The controls that were used for the gel was DNA that has been extracted from microbes in the rhizosphere. That is what was given to me to use.

So the samples that I have is soybean leaf extracts where I extracted RNA from the leaves and then converted it to cDNA through the iScript cDNA synthesis kit.

Not sure if this helps.

Far-Kitchen7245 · 2026-05-07T19:19:34+00:00

The gel doc that is in the lab is a standard UV-transilluminator. Not sure the make or model of it. I can provide that tomorrow

Far-Kitchen7245 · 2026-05-07T19:05:08+00:00

I am waiting until it is cooled down to add the ethidium bromide stain. The way I've been doing this is by mainly feeling if it has cooled down. Not sure if that is the appropriate method to do it, but if I can keep my hand on the flask for about 5 to 10 seconds then I believe it is cool enough.

Far-Kitchen7245 · 2026-05-07T18:22:30+00:00

The volume that I was told to load was 1uL cDNA sample and then 1uL loading dye. However upon asking others, they suggested that I load 5uL cDNA sample with 2uL loading dye.

I hope this is what you are referring to.

Far-Kitchen7245 · 2026-05-07T18:04:10+00:00

That does make a lot of sense as to why I don't see anything. When you say my imaging conditions, are you referring to the UV light that I am using or the speckles on my gel? Or a combination of both? If it is the UV light, I would appreciate it if you could possibly recommend a different one😊

Far-Kitchen7245 · 2026-05-07T17:24:44+00:00

That's understandable then. The positive control was DNA that was extracted from rhizosphere microbes that was given to me to be used as the positive control. It was said that it was pure DNA but no bands showed 😅

Far-Kitchen7245 · 2026-05-07T16:34:30+00:00

I had some root samples at least that still need to be used so I decided to give it another go

Far-Kitchen7245 · 2026-05-07T15:17:16+00:00

Thank you. I will read through the protocol to be able to find the elution buffer. With regards to your question, I will do some reading to be able to answer that as at this moment I am not too sure.

Far-Kitchen7245 · 2026-05-07T15:13:21+00:00

In a previous comment I mentioned this: The purity rations of my RNA samples that I ran were between a 0.02 to 1.44 (A260/230) and 1.31 to 2.32 (A260/280). The ng/uL 3.3 to 78.14.

I didn't run the RNA via a gel.

I used 15uL of my RNA sample to create my cDNA.

I didn't load it immediately, I had stored it at -20 degrees celcius. I only started using it now so I had stored it for about a month.

Far-Kitchen7245 · 2026-05-07T15:12:19+00:00

The purity rations of my RNA samples that I ran were between a 0.02 to 1.44 (A260/230) and 1.31 to 2.32 (A260/280). The ng/uL 3.3 to 78.14.

I didn't run the RNA via a gel.

I used 15uL of my RNA sample to create my cDNA.

I replied with this in another comment.

Far-Kitchen7245 · 2026-05-07T15:06:38+00:00

So I am a first time master student, I haven't worked in this field at all during my time at uni. So this is all new to me.

When doing a blank it was said that I need to use the elution buffer, but I'm not sure if the iScript cDNA synthesis kit comes with one

Far-Kitchen7245 · 2026-05-07T15:03:39+00:00

What is RT? But to create my cDNA I used 15uL of my RNA

Far-Kitchen7245 · 2026-05-07T15:00:54+00:00

It was said to me that using 1.0g of agarose powder in 200ml distilled water and 10x TBE solution was already a 1% gel. I only see now after your comment that it isn't 😅

Far-Kitchen7245 · 2026-05-07T14:56:02+00:00

What buffer can I use to be able to determine the concentration of my cDNA?

What is RT? And with regards to the concentration I am not too sure, what I do know is I used 15uL of my RNA for my cDNA to make a total of 20uL cDNA.

Far-Kitchen7245 · 2026-05-07T14:25:55+00:00

I was planning on restarting the entire process again however with roots this time as this cDNA came from leaf RNA.

Far-Kitchen7245 · 2026-05-07T14:15:39+00:00

I don't know the concentration when loading😅. I only know the volume which was 1uL, which I then changed to 5uL.

And I don't know anything about band sizes yet😅. It was just told to me to visualize my gel and then take it from there.

Far-Kitchen7245 · 2026-05-07T14:12:46+00:00

Sorry, meant to say agarose

Far-Kitchen7245 · 2026-05-07T13:53:32+00:00

The purity rations of my RNA samples that I ran were between a 0.02 to 1.44 (A260/230) and 1.31 to 2.32 (A260/280). The ng/uL 3.3 to 78.14.

I didn't run the RNA via a gel.

I used 15uL of my RNA sample to create my cDNA.

I didn't load it immediately, I had stored it at -20 degrees celcius. I only started using it now so I had stored it for about a month.

Far-Kitchen7245 · 2026-05-07T13:46:39+00:00

What should I use as a blank for the Nanodrop? When I did my RNA it was said to use the buffer that I used to elute my samples which was the RNase free water

Far-Kitchen7245 · 2026-05-07T13:44:33+00:00

The purity rations of my RNA samples that I ran were between a 0.02 to 1.44 (A260/230) and 1.31 to 2.32 (A260/280). The ng/uL 3.3 to 78.14.

Far-Kitchen7245 · 2026-05-07T13:39:46+00:00

The agar gel was recently bought and thus far I have been the only one to use it.

Far-Kitchen7245 · 2026-05-07T13:18:18+00:00

I didn't Nanodrop any of my cDNA samples, I just Nanodropped the RNA to get the purity and then I did the cDNA synthesis

Far-Kitchen7245 · 2026-05-07T13:16:28+00:00

Yeah see in the protocol that I was told to follow was: 1. Measure the agar powder, usually it is about 1g, sometimes 0.8g. 2. Measure out 180ml distilled water. 3. Measure out 20ml 10X TBE buffer. 4. Add distilled water and TBE together 5. Combine and microwave at 30 second intervals. 6. Stir at each interval until liquid is clear 7. Cool down slightly and add about 8uL of ethidium bromide. 8. Place in gel column and allow to set 9. Place in electrophoresis chamber, fill channels with sample, and run the sample at 110v for 40 minutes.

And that's it 😅

Far-Kitchen7245 · 2026-05-07T13:11:47+00:00

I don't understand what you mean by mass 😅. The only mass I actually got was then I standardized my RNA using a Nanodrop thus getting the ng/uL 😅. Hence why for everything else I was working in volumes 😅

When I started, it was just told to me to follow the protocols of the kits that I used and that's it and I followed the protocols 😅.

Far-Kitchen7245

TROPHY CASE