Is it western blot?
Not a super high yield topic but I missed a question on my last FL because I couldn’t differentiate.
The mnemonic is SNOW DROP
S(outhern) corresponds to D(NA).
N(orthern) corresponds to R(NA)
O place holder O
W(estern) corresponds to P(rotein).
Easy mnemonic so you don’t miss free points!
Happy studying 🥰
I have always been confused between these different blots. Can someone please explain the difference and the mechanism for all three blots.
Thank you!!!
Okay it's not mine, but I found it and would like to share:
SNoW DRoP (snow drop)
S D : southern/DNA
N R : northern/RNA
o o
W P : western/protein
edit: I have been informed that this is a popular mnemonic. I'll keep this post up for the dumbasses like myself who didn't know about it
Hi lab rats,
I'm trying to do my first ever Northern blot. I'm not a molecular biologist by training so I'm not sure what I'm doing wrong to not get any signal.
I'm looking for a small, noncoding RNA in e. coli cells. The RNA I'm looking for is only 77bp long and forms two hairpin structures. I'm using a probe that has been previously published; I don't have radioactivity training so I'm using biotinylated probes and using streptavidin-HRP to detect (this is how a previous paper did their Northerns, and their results look fine). I used the hot phenol method to do the RNA extractions and I think I got quite a lot of RNA, something around 2.5-4 ug/uL. I DNase treated and did a phenol:chloroform extraction after the DNase treatment, so I'm moderately confident that there is little to no DNA in my samples.
I ran my RNA samples (5ug total per lane) on a 15% TBE-Urea gel (the mini-Protean ones from Bio-Rad) for 2 hours at 75V. I checked the quality of the RNA by ethidium bromide staining and there was a large, very bright smear in each lane that I loaded, so I think the gel ran okay. I did a semi-dry transfer in 0.5x TBE buffer; constant 200mA for 1 hour. I forgot to stain the gel post-transfer (I will do this next time) but there was blue dye on the membrane so I think at least some of the RNA transferred. I used Thermo's UltraHyb solution to do the hybridization. I hybridized the probe at a concentration of 100ng/mL in the UltraHyb buffer, for 15 hours at 42 with gentle shaking (don't have a hyb oven). I used Thermo's chemiluminescent nucleic acid detection module to do the detection (in a nutshell: first you block with blocking buffer, add the blocking buffer with streptavidin-HRP, wash four times, then add luminol/enhancer solution and image).
But: nothing. No signal! There was some signal on the very edges of the membrane, but nothing in the middle.
Northern experts: help???
How do you test whether unknown bands are splice variants or mRNA products of 3 different genes?
They both have to do with detecting RNA but is one used over the other in certain conditions?
Anyone feel like tackling a mystery today? :)
For the last several days, Northern blots have been failing for everyone in my lab. They all look more or less the same: the 50 bp mark of the ladder is fairly strong, but signal on the rest of the blot (both ladder and samples) is almost nonexistent. Since the last successful blot on Thursday, every subsequent blot has failed in the same way. This is happening across multiple people, so it must be a problem with equipment or reagents. We think it's a problem with the transfer, but also aren't 100% sure on that, since we still see the expected dark/light blue bands of our loading buffer on the membrane after we transfer.
Between the last successful blot and the first failed one, two things were different: a new batch of TBE, and a new box of membrane. We assumed the TBE was somehow wrong and we changed it immediately, but the problem continued! The other components seem fine:
-We have two transfer tanks/power supplies we've been using, so they can't both have broken at once
-Our imager still works correctly for older blots that were made before the problems started
-No other buffers, etc. were changed between blots.
It seems like the only thing left is the membrane. So my questions is, has anyone ever encountered a "bad batch" of membrane before? Or, is there something else we are overlooking that might be causing our problems? We instead to test a new box of membrane tomorrow, but it would be good to hear other tips in case that doesn't work out.
Thank you!!
Update: Turns out it was the membranes! We're seeing about getting them replaced. Thanks for all the tips :)
I totally forgot a step in my protocol, a pretty important one at that, and don't know if there's a way to recover. I have a feeling I'm shit out of luck and should just start over...
Hi all
I am currently doing qPCR work but I am curious for those of you who have experience running all 3 different techniques for gene expression. What were the cost like for each of them? What would be the most cost effective way to run gene expression experiments?
" the Gene has one intron that is 500 nucleotides long. after its intron is removed from the pre-mRNA, the mRNA transcript is 1000 nucleotides in length. You work with diploid somatic cells, therefore you have two copies of the gene"..
in lane 4: " a homozygote with a mutation in the middle of the coding sequence that results in the premature stop codon" So since it is a homozygote it would be marked with a thick band and around the 750 nucleotides mark? Just trying to make sure im doing this right thank you.
If I clean it will it still work or do I have to start all over again? To be clear it was dropped after the electrotransfer.
E: I'll update you all tomorrow with the results tomorrow!
So I'm currently infecting mammalian cells with virus and harvesting the cells for RNA extraction, followed by a northern to detect differences in viral RNA. I'm looking for a particular non - coding RNA that's relatively big (0.5kb).
I have never done a northern before so I'm wondering what kind of controls I need to include? I've read some papers that probed for GAPDH or rRNA as internal controls but I don't really understand why, other than it 'normalizing' the data.
So my question is what kind of controls should I consider for a nothern blot when I'm probing for noncoding viral RNA?
Edit: Any extra advice for not screwing up northerns would be amazing too!
This is homework help. I am stuck on a weird assignment question.
It relates to this paper and the question reads: >In Figure 1F, why is the scm-1 RNA abundance decreased? Hint- think laterally.
I took a screen cap of the figure in question for those of you without access to the journal.
According to someone who has done this course before, the answer is not along the lines of polymerase skipping over the stop codon occasionally, or a shorter version of the RNA being produced.
I feel like there is a clever and creative answer that is actually super obvious, but it is eluding me. The question is not even worth any significant marks - it is just bugging me that I can't think of anything.
Any thoughts?
Update 1: Thanks for your responses! I was able to find a reasonable way by sequencing the cDNA I get from the two RNAs and comparing them for similarities.
I am trying to look at plant gene expression using northern blots, but it has not been successive for me. After exposing to film, I see the RNA bands clearly, but it is the background that is darkened. Almost like the probe is binding to everything but RNA. Does anyone have any suggestion where the problem happened? Dot blots worked fine, which makes me think it was in the membrane preparation.
I've been doing Northern Blots recenty, and I know that RNA is usually crosslinked to a nylon membrane by UV irradiation. But I did not find any information about the chemistry that is happening during the irradiation. Does anyone know this, or find a paper about this?
so far i get southern and northern blotting.. . buttt can someone plss explain these techniques and the objectives of them to me like i'm 5 years old?
From what i understand:
southern=DNA so you would look for changes in the DNA itself
Northern=RNA so you would look at gene expression
Western=protein and look at changes in protein activity?
Did i get these right? Is there more we need to know about these types of blots?
