3 ms·

For doing mutagenesis on plasmids, you don't even need gibson. Overlapped primers do some weird circularization during PCR, so for point mutations you don't eve

by koeng 6y ago

For doing mutagenesis on plasmids, you don't even need gibson. Overlapped primers do some weird circularization during PCR, so for point mutations you don't even need gibson mix. I'm personally a big fan of synthesis + GoldenGate for building genetic circuits. Gibson, IMO, destroys reproduciblity (how many different PCR kits are there?) and leaning towards Gibson assembly over enzymatic assembly methods (GoldenGate, maybe even BioBricks) makes biotech an Ivory-Tower exercise, since not everyone has access to oligos.

dnautics 6y ago
> since not everyone has access to oligos I don't know about that. I've bought 50-mer oligos at $20 a pop to do a 6-part assembly in my garage (I made a strain of yeast that generates cannabinoids). DNA oligos "basically last forever" if you handle them relatively correctly, even in a terrible defrost-cycling consumer freezer. Enzymes, especially specialty ones, not rock solid ones like modern TAQ, have a very short halflife in your freezer, especially if your freezer is consumer-grade. > how many different PCR kits are there? Yeah, just buy NEB. Seriously. Not worth screwing around with companies that have lower QC standards.
- koeng 6y ago
  NEB doesn’t ship residential, and there are many parts of the world that you can’t buy $20 oligos from. I like doing pretty complicated builds, and so I’d go bankrupt pretty much immediately because I don’t just build one thing.
  - dnautics 6y ago
    I've gotten neb to ship residential...
vikramkr 6y ago
How is gibson ivory tower? It's literally just based on overlapping sequences. And how is it hard to reproduce? You select out colonies and verify with sanger. These are not things that are inaccessible to most people, especially if you live near a DIY bio hacker space, and if you get involved in the biohacker community you'll get a sense of what providers ship to residential address, where to get certain equiptment, and so on. Just googling will get you information that can help you figure out how to get what you need. https://medium.com/@ThatMrE/a-guide-to-diybio-updated-2019-abd0956cdf74 https://medium.com/@ThatMrE/a-guide-to-diybio-updated-2019-a...
- koeng 6y ago
  Reordering of oligos is expensive, which makes people not reproduce. Sanger sequencing is pretty expensive ($5 sure, but over 5kb on 3 different constructs becomes pricey). PCR machines also ain’t that cheap (when you’re a noob and don’t know what to buy), especially when you need to do more than 8 PCRs at a time. In theory, works in practice, and in practice, works well in theory. Even for experienced DIYbio folks, cloning isn’t just 1,2,3. Also, PCRs themselves aren’t very reproducible. It’s not about the overlapping sequence.

by koeng 6y ago

dnautics 6y ago

> since not everyone has access to oligos I don't know about that. I've bought 50-mer oligos at $20 a pop to do a 6-part assembly in my garage (I made a strain of yeast that generates cannabinoids). DNA oligos "basically last forever" if you handle them relatively correctly, even in a terrible defrost-cycling consumer freezer. Enzymes, especially specialty ones, not rock solid ones like modern TAQ, have a very short halflife in your freezer, especially if your freezer is consumer-grade. > how many different PCR kits are there? Yeah, just buy NEB. Seriously. Not worth screwing around with companies that have lower QC standards.

koeng 6y ago
NEB doesn’t ship residential, and there are many parts of the world that you can’t buy $20 oligos from. I like doing pretty complicated builds, and so I’d go bankrupt pretty much immediately because I don’t just build one thing.
- dnautics 6y ago
  I've gotten neb to ship residential...

vikramkr 6y ago

How is gibson ivory tower? It's literally just based on overlapping sequences. And how is it hard to reproduce? You select out colonies and verify with sanger. These are not things that are inaccessible to most people, especially if you live near a DIY bio hacker space, and if you get involved in the biohacker community you'll get a sense of what providers ship to residential address, where to get certain equiptment, and so on. Just googling will get you information that can help you figure out how to get what you need. https://medium.com/@ThatMrE/a-guide-to-diybio-updated-2019-abd0956cdf74 https://medium.com/@ThatMrE/a-guide-to-diybio-updated-2019-a...

koeng 6y ago
Reordering of oligos is expensive, which makes people not reproduce. Sanger sequencing is pretty expensive ($5 sure, but over 5kb on 3 different constructs becomes pricey). PCR machines also ain’t that cheap (when you’re a noob and don’t know what to buy), especially when you need to do more than 8 PCRs at a time. In theory, works in practice, and in practice, works well in theory. Even for experienced DIYbio folks, cloning isn’t just 1,2,3. Also, PCRs themselves aren’t very reproducible. It’s not about the overlapping sequence.