5 ms·

Would this be easy to do yourself if you were able to find (or make) a PCR machine?

by voldacar 7y ago

mjfl 7y ago
You would also need a microcentrifuge and pipettes and stuff, but yes.
- gravelc 7y ago
  You need a quantitative real-time PCR machine, not just a PCR machine. Not cheap. EDIT: You can't just put this on a gel. Something that comes up at 35 cycles (likely negative - needs re-running) looks exactly the same as something that comes up at 15 cycles (likely positive). The quantitative part is important. 2nd EDIT: it uses dual-labelled probe - end point PCR is absolutely not good enough.
  - copperx 7y ago
    Why is that? Is it really impossible to do the test on a simple PCR machine, or is it just too slow?
    - gravelc 7y ago
      pPCR is quantitative - measures fluorescence. End point PCR just measures whether you have the sequence there or not. It's slower, more work, and prone to false positives (see edit above).
  - mjfl 7y ago
    Or you could just run the PCR product on a gel for much cheaper... Edit: A gel gives you the ability to select for size as well, which a qpcr machine does not. Not sure why I have to reply in the form of edits.
  - jevinskie 7y ago
    I saw a whole kit including laptop, software, and license for $3,500 buy-it-now on eBay last week.
  - hcknwscommenter 7y ago
    Totally wrong. Take a sample out in 15 cycles. Take a sample in 30. Run a gel. Compare. Done. Most of the issues you think exist are because an RT pcr machine cannot distinguish between primer-dimer and pcr product. A gel can distinguish.
    - gravelc 7y ago
      This argument is pointless as the assay relies on a dual-labelled probe. Look up how that works [1]. Anyway, an end-point PCR is not quantitative. You could do semi-quantitative on a gel, but it's a rubbish technique that was rightfully forgotten years ago, as it requires a lot of optimisation and has very little reproducibility. [1] https://www.sigmaaldrich.com/technical-documents/articles/biology/dual-labeled-probes.html https://www.sigmaaldrich.com/technical-documents/articles/bi...
      - test7777 7y ago
        you really think anyone cares how much of the corona you got in the particular blob of snot you gave for testing?
        hcknwscommenter 7y ago
        You deserve a 1000 upvotes for your succinct and accurate comment.
      - hcknwscommenter 7y ago
        You are totally wrong. Your comment is dangerous misinformation. NO coronavirus assay requires quantitation.
      - hcknwscommenter 7y ago
        You seem to miss the point entirely. The point is that the assay doesn't NEED or REQUIRE dual labelled probes at all. The only need for quantification at all is to avoid mistaking signal for spurious noise (e.g., primer dimer). The gel provides both semi-quantification and provides amplicon size, which massively increases specificity of an end-point version of the assay. It is perfectly adequate. The larger point is that there are many ways the test could have been designed around any possible combination of supply roadblocks (no DNA extraction reagent, choose phenol/chloroform; no cotton swabs, use paper; I could go on and on), so there are no excuses as to the massive and unwarranted and fatal delay in rolling out comprehensive testing in the U.S. Sure some work arounds are less desirable, but the alternative we have chosen is to fail a number (~>5,000) of people who didn't have to die by letting this thing spread without adequate testing.
  - test7777 7y ago
    on a gel you see the size, something coming up with exactly the right size even at 35 cycles indicates that there was at least some of what you designed the primers on in the sample. if you want higher certainty, with three primers you can get two different sizes ruling out any doubt. with the rtpcr machine readout which only gives you the signal corresponding to how much was amplified over time you need tricks like the dual labelling.
joshuamcginnis 7y ago
FYI, the same core equipment and supplies will also allow you to do dna-based lab work, such as crispr/cas and fingerprinting for phylogeny, at home. If you're clever, you can replicate the same cutting-edge work step-by-step from published research papers and then use that knowledge to run your own experiments. I'm currently working on adding the gene for bioilluminescence to p. cubensis fungi. If I can successfully breed offspring with the desire genotype, I hope to sell the novel spores and use those funds to invest in a variety of altruistic research like individualized DNA-based cancer treatment.
- TheSpiceIsLife 7y ago
  Governments could open source all patents and design specifications for all relevant technology.
- voldacar 7y ago
  How hard is it to get started doing stuff like this? Do you need thousands of dollars of lab equipment or is the entry barrier relatively low? Making your own bioluminescent genetically modified organisms sounds really neat
  - subroutine 7y ago
    You need a set of pipettes, a thermal cycler (i.e. https://www.ebay.com/b/PCR-Thermal-Cyclers/185231/bn_115383501 https://www.ebay.com/b/PCR-Thermal-Cyclers/185231/bn_1153835... ), some consumables (i.e. pipette tips, vials, gels), reaction reagents (listed by the top level comment), a centrifuge, and probably some other things I'm forgetting. For some reactions you are much better off using a quantitative PCR machine but those are quite expensive. If you just want to use molecular biology to make things glow see: https://www.minipcr.com/products/p51-molecular-glow-lab/ https://www.minipcr.com/products/p51-molecular-glow-lab/
    - voldacar 7y ago
      thanks so much, this will be a fun rabbit hole i think
asdff 7y ago
You can make your own pcr machine with thermometers, 3 pots of water on your stove, and a stop watch
hcknwscommenter 7y ago
Yes. You can buy a PCR machine for about $1,000 US. You can build your own agarose gel box for about $100. THe power supply might be tricky, but probably not. The CDC has published all of the primer and probe sequences. It is trivial to run the assay yourself if you buy the PCR machine and a few water baths for the RT and DNAse reactions.
- juuul 7y ago
  If you aren't worried about safety then you can use e.g. a full bridge rectifier hooked up to 110 V AC to get a usable (but shitty) agarose gel power supply. I really don't recommend this but it does show how easy it can be.

Would this be easy to do yourself if you were able to find (or make) a PCR machine?

mjfl 7y ago

You would also need a microcentrifuge and pipettes and stuff, but yes.

gravelc 7y ago
You need a quantitative real-time PCR machine, not just a PCR machine. Not cheap. EDIT: You can't just put this on a gel. Something that comes up at 35 cycles (likely negative - needs re-running) looks exactly the same as something that comes up at 15 cycles (likely positive). The quantitative part is important. 2nd EDIT: it uses dual-labelled probe - end point PCR is absolutely not good enough.
- copperx 7y ago
  Why is that? Is it really impossible to do the test on a simple PCR machine, or is it just too slow?
  - gravelc 7y ago
    pPCR is quantitative - measures fluorescence. End point PCR just measures whether you have the sequence there or not. It's slower, more work, and prone to false positives (see edit above).
- mjfl 7y ago
  Or you could just run the PCR product on a gel for much cheaper... Edit: A gel gives you the ability to select for size as well, which a qpcr machine does not. Not sure why I have to reply in the form of edits.
- jevinskie 7y ago
  I saw a whole kit including laptop, software, and license for $3,500 buy-it-now on eBay last week.
- hcknwscommenter 7y ago
  Totally wrong. Take a sample out in 15 cycles. Take a sample in 30. Run a gel. Compare. Done. Most of the issues you think exist are because an RT pcr machine cannot distinguish between primer-dimer and pcr product. A gel can distinguish.
  - gravelc 7y ago
    This argument is pointless as the assay relies on a dual-labelled probe. Look up how that works [1]. Anyway, an end-point PCR is not quantitative. You could do semi-quantitative on a gel, but it's a rubbish technique that was rightfully forgotten years ago, as it requires a lot of optimisation and has very little reproducibility. [1] https://www.sigmaaldrich.com/technical-documents/articles/biology/dual-labeled-probes.html https://www.sigmaaldrich.com/technical-documents/articles/bi...
    - test7777 7y ago
      you really think anyone cares how much of the corona you got in the particular blob of snot you gave for testing?
      - hcknwscommenter 7y ago
        You deserve a 1000 upvotes for your succinct and accurate comment.
    - hcknwscommenter 7y ago
      You are totally wrong. Your comment is dangerous misinformation. NO coronavirus assay requires quantitation.
    - hcknwscommenter 7y ago
      You seem to miss the point entirely. The point is that the assay doesn't NEED or REQUIRE dual labelled probes at all. The only need for quantification at all is to avoid mistaking signal for spurious noise (e.g., primer dimer). The gel provides both semi-quantification and provides amplicon size, which massively increases specificity of an end-point version of the assay. It is perfectly adequate. The larger point is that there are many ways the test could have been designed around any possible combination of supply roadblocks (no DNA extraction reagent, choose phenol/chloroform; no cotton swabs, use paper; I could go on and on), so there are no excuses as to the massive and unwarranted and fatal delay in rolling out comprehensive testing in the U.S. Sure some work arounds are less desirable, but the alternative we have chosen is to fail a number (~>5,000) of people who didn't have to die by letting this thing spread without adequate testing.
- test7777 7y ago
  on a gel you see the size, something coming up with exactly the right size even at 35 cycles indicates that there was at least some of what you designed the primers on in the sample. if you want higher certainty, with three primers you can get two different sizes ruling out any doubt. with the rtpcr machine readout which only gives you the signal corresponding to how much was amplified over time you need tricks like the dual labelling.

joshuamcginnis 7y ago

FYI, the same core equipment and supplies will also allow you to do dna-based lab work, such as crispr/cas and fingerprinting for phylogeny, at home. If you're clever, you can replicate the same cutting-edge work step-by-step from published research papers and then use that knowledge to run your own experiments. I'm currently working on adding the gene for bioilluminescence to p. cubensis fungi. If I can successfully breed offspring with the desire genotype, I hope to sell the novel spores and use those funds to invest in a variety of altruistic research like individualized DNA-based cancer treatment.

TheSpiceIsLife 7y ago
Governments could open source all patents and design specifications for all relevant technology.
voldacar 7y ago
How hard is it to get started doing stuff like this? Do you need thousands of dollars of lab equipment or is the entry barrier relatively low? Making your own bioluminescent genetically modified organisms sounds really neat
- subroutine 7y ago
  You need a set of pipettes, a thermal cycler (i.e. https://www.ebay.com/b/PCR-Thermal-Cyclers/185231/bn_115383501 https://www.ebay.com/b/PCR-Thermal-Cyclers/185231/bn_1153835... ), some consumables (i.e. pipette tips, vials, gels), reaction reagents (listed by the top level comment), a centrifuge, and probably some other things I'm forgetting. For some reactions you are much better off using a quantitative PCR machine but those are quite expensive. If you just want to use molecular biology to make things glow see: https://www.minipcr.com/products/p51-molecular-glow-lab/ https://www.minipcr.com/products/p51-molecular-glow-lab/
  - voldacar 7y ago
    thanks so much, this will be a fun rabbit hole i think

asdff 7y ago

You can make your own pcr machine with thermometers, 3 pots of water on your stove, and a stop watch

hcknwscommenter 7y ago

Yes. You can buy a PCR machine for about $1,000 US. You can build your own agarose gel box for about $100. THe power supply might be tricky, but probably not. The CDC has published all of the primer and probe sequences. It is trivial to run the assay yourself if you buy the PCR machine and a few water baths for the RT and DNAse reactions.

juuul 7y ago
If you aren't worried about safety then you can use e.g. a full bridge rectifier hooked up to 110 V AC to get a usable (but shitty) agarose gel power supply. I really don't recommend this but it does show how easy it can be.