Gel electrophoresis operates as an electrolytic cell correct? then why do negatively charged molecules at the cathode travel down to the positive anode? (- to +). Isn't that indictive of a galvanic cell? from my understanding electrolytic cell operates on the basis of bringing positive potential to negative potential (+ to -)
The RA in our lab has recently gone on leave, so the genotyping of our mice has been split amongst a few graduate students. We did run through everything with the RA, but now that she's gone we've been having a lot of problems.
We first extracted the samples and ran the PCR/gels last Wednesday, and saw some banding, but a lot of it was quite faint (image 1).
When we re-ran it, using the same working solutions of reagents but making extra sure to mix the samples, we saw no amplification at all (only "primer-dimers"(?)) (image 2).
We then re-made new working solutions of primers from stock and new buffer for the gel, but again saw no amplification when we ran it (image 3 - final well here was to test for contamination in one of the reagents for another line).
Then, yesterday, we re-ran it again, with new buffer, new primer solutions, and we also extracted and ran some new samples, as well (image 4). The first gel, with the "old" samples, had only some very faint bands (like the first time we ran it, in image 1), but the other two gels were pretty good. We thought potentially the "old" samples had degraded, and maybe that was why we weren't getting good results, but four samples from the "old" batch were overflow on the third gel, where they turned out quite well. All of the gels were made in a batch together, as was the buffer solution they were run with, and all of the samples underwent PCR in the same machine with the same mastermix of primers, red extract, etc.
This morning I tried to re-run some of the newer samples that weren't quite clear yesterday, along with some of the "old" batch, using the same working solutions of primers and buffer we made the day before (image 5). I saw basically no amplification again, like we previously saw in image 2 and image 3. I made sure I was vortexing the necessary things as I was adding them, and paid close attention to the volumes/concentrations o
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How much do I add to where it’s covering the gel or just touching the gel?
Hey guys,
Tldr: our gels look like this even after months of RCA and we still can't figure out what's causing smear city
I'm.not sure where else to ask this or where to go, so if this is against the rules or etiquette, then mods, take it down. If not, then please read on. I want to be as vague as possible because I'm super anxious about our lab's policies on discussing methods outside of work, but I think this will do.
We are a PGx Lab and our SOP for detecting the a particular phenotype of a particular gene calls for good old fashioned gel electrophoresis.
Now, about 4 months ago, our bands on our gels were are clear and as crisp as day. Not a single smudge or smear. But suddenly, they started smearing, out of nowhere, into unreadable bands. This pushed back our TAT and really harmed our relations with a few clients and patients.
First, we thought it was our forward/reverse primers from one manufacturer. I think it was ThermoFischer. So we switched to Eurofin and got fresh F/R primers. That yielded no improvement.
We tried switching out to brand new clean buffer for our gel box. No improvement. We increased our MSP1 digestive enzyme from 0.2uL to 0.5uL. nothing. 0.2uL, nothing.
We aren't convinced there's any contamination, but I'm going to try to switch to a new container for our 1X TBD Buffer when I make our agarose tomorrow morning. So maybe that'll help?
I'm trying to remember everything off the top of my head; I'm asking this representing my supervisor and team, so bear with me in regards to a few nuanced details, but I can fare well with most questions you can ask. If anything, I'll get the info as soon as I can.
How should we approach this issue? The smearing severely hurts, not only us, but the poor patient and the practices.
Please let me know if there are any other details I can provide!
Help us, fell biologist-kenobis, you're all are our only hope!
Gel electrophoresis is a technique that separates macromolecules in an electrical field. It is a common method in Molecular biology to separate DNA, RNA and proteins from mixtures according to their molecular sizes. SDS Page is a type of gel electrophoresis which is used to separate proteins from a protein mixture based on their sizes. Gel electrophoresis is a term used to refer to the normal technique applied for DNA, RNA, and protein separation while SDS Page is one type of gel electrophoresis. This is the key difference between gel electrophoresis and SDS Page.
Gel Run
It can be performed in a horizontal or vertical manner.
SDS Page always runs vertically.
Basis for Separation
Gel electrophoresis: Separation occurs according to the charge and size.
SDS Page: Protein separation occurs according to the mass and charge.
Resolution
Agarose gel electrophoresis has low resolution and polyacrylamide gel electrophoresis has a higher resolution
SDS Page has a better resolution.
Denaturation
Gel electrophoresis includes both denaturing and non-denaturing techniques.
SDS Page denatures proteins prior to separation.
Hi bio nerds!
I was stuck on the question for ages and my teacher's explanation wasn't that helpful because I can't seem to relate the two diagrams here, but her explanation gave me a better direction.
- what even is the first diagram for? is it just when the recognition sites appear in the dna strand?
- why are there n+1 bands for n appearances of the recognition sites on the second diagram?
- what is going on hahah!?!?
Thanks to whoever has amazing explanation skills to explain this to me:)
https://preview.redd.it/szwdjizif7j71.png?width=594&format=png&auto=webp&s=8132bf2dd1dc044d8d7909547c45837b639f2c84
Hello all, I've tried finding a good explanation of the difference between the different types of gel electrophoresis (I.e. Native, SDS, reducing, non-reducing, etc.), but for some reason my confusion has not cleared up ye. I have a rough overview of how each one works.
Native just keeps proteins in their normal shape, does not disrupt them at all and separates based on size and charge, and you can get your protein again at the end.
SDS-PAGE adds a detergent which gives it a strong negative charge so you're only separating based on size.
Reducing disrupts disulfide bonds between subunits, so they will appear as individual bands corresponding to each monomer. If it is a homodimer or homotetramer where all the monomers are identical, it will still just appear as one band, but if it is a heterodimer, etc. then it will appear as different bands.
Non-reducing I have no idea other than that it doesn't do what reducing does lol
I'm still a little confused on what level of protein structure is affected by each one and what non-reducing does. Appreciate any and all help and thank you in advance!
I've been looking for a software to quantify the bands on electrophoresis gels,
Any suggestions?
I routinely do gel electrophoresis and have never had this issue.
I ran a gel (1% agarose, pre-stained with 10 000X SYBR safe) and used a blue light transilluminator to detect bands. But last night I could not see any bands, including the ladder. I repeated a couple of times but had the same issue. For my last gel, I tried a post-stain method and that fixed the issue. But my question is: why after almost two years of doing this, I have only encountered this issue now? I cannot think of what I have done differently. Not to mention other people in lab did not have that issue yesterday (and they also pre-stain).
https://youtu.be/I0bgA9mu66o
So a few things I am sure of. Anode is the site of oxidation and the cathode is the site of reduction. I thought electrons always flowed from anode to cathode, but as I’m reading anode/cathode explanations on this subreddit, I’ve seen some people say that the electrons flow from cathode to anode in electrolytic cells and others say they ALWAYS flow anode to cathode. So which direction do electrons flow?? Can someone also explain the charges of the anode and cathode in voltaic cells, electrolytic cells, and gel electrophoresis (which I know is considered an electrolytic cell)? Super confused on all this so a very simple explanation would be much appreciated lolol
Hello there,
I have one question regarding gel electrophoresis. From the gel photo, how can you determine the type of mutation? I mean if the mutation is deletion, insertion, or a point mutation?
Thanks in advance
Hi all,
I've been taking some notes on the Lab Techniques PDF so I thought I'd share with you my super concise notes on the first portion: Gel electrophoresis. Lemme know if you'd like me to keep sharing my notes
Also for anyone wondering I used the app notability on the iPad!
