Can you do an acrylamide gel? If not probably need some other technique to separate them out. Is the goal to cut the band or just see difference between lanes?
Very doable! Any chance you can design specific PCR primers where one lands in that 35 bp region? Or as you've probably already checked if there's a unique cut site there
There was this coding competition called "ugliest code" or something where the most roundabout convoluted way to solve a problem won. So hilariously awesome.
How many samples are we talking? Plasmidsaurus long read Seq should work to quantify % of amplicons of each length. But 15 bucks a pop may not scale well if this needs to be run on tons of samples
Its possible you could see the difference without probes and just a melt curve at the end, but that would probably depend on the nature of the inserted bases.
If they have >100 samples, it's probably worth the work to figure out qpcr. Less than that, just sequence
Or use the sequence difference to design internal primers that for a second PCR round that will only produce a shorter band in the presence of 1 of the 2 products.
6% acrylamide 30:1 bis:acrylamide in 1XTBE. Run it on a long sequencing gel for a long time. Rain-x the notched plate so when you split the plates, the gel sticks to only 1 plate. Then soak it in stain of choice, visualize, cut out the band of interest and electroelute to purify.
First recommendation: don’t do that. Is is absolutely necessary to amplify that fragment or can they redesign the PCR to have a shorter amplicon? What is the product being used for afterwards - do you need to go on to clone the full thing?
If it’s just visualization, redesign the primers to make 35 bp >5% (ideally >10%) of length and run an agarose gel. Primers are cheaper than restriction enzymes and agarose gel can be reused many times over.
Do you need these to be intact? And do they have similar sequences? If they have some sequence differences, you could melt the DNA and use a biotinylated primer to selectively isolate one of the PCR products.
IDT sells biotinylated primers pretty inexpensively and if you had a way to uniquely target each band you could do the pulldown with some magnetic beads pretty quickly. But plasmidsaurus has sequencing services that cost $15-30 a pop and you could sequence end to end.
Agarose wont have the resolution. Challenge even with a 6% long sequencing gel, will require a very long run and a thin gel which is tricky to stain - probably using silver stain.
Basically it the same primers but different alleles will give either 1) a 1035bp product or 2) both a 1035bp or 1000bp product. They're trying to visualize 1 vs 2 bands in an individual's PCR product(s).
Best bet is to cut using a restriction enzyme so that the size difference is more than 10% of the fragment size, then use appropriate agarose percentage gel. So 270bo, 300bp ish, 2% will be fine.
Quick amplicon prep on an @nanoporetech.com sequencer. Loads of service providers out there doing amplicons - @plasmidsaurus.bsky.social do them very very cheaply https://plasmidsaurus.com/
If that isn't possible, I would cut them with an RE that cuts in multiple places such that the variant is inside a smaller (<175 bp) fragment. On a gel (high % agarose?), just the one band will vary.
Tricky - 1% difference is not enough I think. I'd design 1 allele-specific primer, or make products smaller so they can be resolved. Eg: 500 vs 535 might be visible on 2% agarose - making sure to load next to allele 1 product, allele 2 product, & mix 1 & 2 product.
Been thinking about this a lot lately, to come up with cheap screen. Where is you 35 bp difference? Roughly mid fragment? And this indel is only difference?
Cool, great you’re crowd sourcing. I’ve been thinking a lot about T7 endonuclease. if the indel is relatively interior, this enzyme might be the trick. mix two versions, rapidly heat and cool them, then you’ll get them reforming as duplexes within size class and between. T7 cuts at the heteroduplex
Comments
https://www.bio-rad.com/en-us/category/pulsed-field-gel-electrophoresis-systems?ID=47e6e174-af10-4db3-9da8-d9141dee988d
https://www.ncbi.nlm.nih.gov/probe/docs/techdcaps/
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pick unique restriction sites close to the 5' and/or 3' end
if fragments are small, run on TBE PAGE
source: https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/Bulletin_6052B.pdf
If they have >100 samples, it's probably worth the work to figure out qpcr. Less than that, just sequence
Do you have known control samples you can use to test things out? SSCP gel might show up something useful if there aren’t any useful RE sites.
Much more accurate than a gel.
Assumed homozygous or haploid
(I figured there's enough serious answers that I can do a little o chem shitposting)
That's a specific type of sick though