And how does it work in practice? Here’s an overview: * choose a vector * design primers to copy just the regions of the vector and the insert you want to combine - instead of primers that just match what’s already there, you make “Special” primers - have ~20bp overlap - basically you design the primers to start with the end of the other piece, so that when you make a copy you also copy a few of the words that you want to come before it when you staple them together * do PCR - you end up with double-stranded DNA (dsDNA) fragments whose ends are hybrids between the vector & the insert - but you still have 2 pieces... and those pieces are pretty content as is… need to chew their ends up a little to create sticky ends like those you’d get from restriction enzymes (now, instead of cleaving in the middle of DNA, which you want ENDOnucleases for you want to chew from the end so you need EXOnucleases * add another endonuclease, DpnI to selectively degrade any of the original parent vector, as that vector would give you false positives in the selection step because it has the antibiotic resistance gene. DpnI only degrades methylated DNA and methylation (addition of a methyl (-CH₃) group) occurs in bacteria but not PCR, so the PCR-generated DNA is *not* methylated and thus is safe, whereas the parent DNA *is* methylated and thus is at risk so DpnI is able to selectively degrade the parent. more here: bit.ly/reasesvsmtases * purify the products (easy to do with a spin column kit) - this is important - want to remove unused nucleotides because you’re about to add a polymerase again! * add T4 polymerase - polymerase? isn’t that what adds the nucleotides? yes - but proofreading polymerases can also remove them - the 3’-5’ exonuclease activity is important for proofreading because it allows the polymerase to “backtrack.” But since you took away the nucleotide train track pieces, the polymerase can’t build track to go forward, so it gets “bored” and starts going the direction it can go - “backwards,” chewing off the ends as it does so and creating single-stranded (ssDNA) overhangs at the 5’ end. You don’t want it to erase all of your (ok, the thermal cycler’s) hard work - you just want it to chew off enough to give you sticky ends, so you only give it ~10 min to do this * then you stop it by adding a dNTP (DNA letter), but just 1 of the letters - it can add this letter, so it switches to its track-laying (polymerization) mode, but then gets stuck and stalls when it needs to add a different letter * at this point you have sticky ends that can stick together, but when they do so there may be gaps because the different strands have gotten chewed different amounts - if you stitched it “as is” you’d be deleting letters. Instead, you want to fill those letters back in, which will require more than just the stitcher, so instead of adding DNA ligase to stitch up the ends in a test tube, we just stick it in bacteria and have the bacteria stitch it up for us using its own machinery - the bacteria’s homologous repair machinery (part of its DNA maintenance crew) is well equipped to handle cases like these A similar method is GIBSON ASSEMBLY - the PCR part’s basically the same but then things change a bit - instead of relying on one enzyme (T4) to do both the chewing & the polymerizing, it uses 2 separate enzymes - T5 exonuclease & Phusion polymerase. It also uses DNA ligase. Gibson is much more expensive because you’re supplying all these products “in vitro”(in a test tube) in their pure form. We don’t need them to be “pure” - we just stick the partial product into bacteria and have them finish the work - their “impure” forms work great! Gibson can be more efficient, but we’ve had great success with SLIC Don’t confuse “Gibson” with “Golden Gate” which uses PCR to add on restriction enzyme sites then uses those restriction enzyme sites to cut & paste (like copy but add cut sites when you copy -> then cut & paste) and don’t confuse “Golden Gate” with “Gateway” which uses λ integrase - a whole different mechanism Whatever method you choose, you still need to make sure it worked, same as with restriction cloning. We can use the same techniques to check if a gene we’re interested in got inserted into the plasmid - techniques like diagnostic digest, blue-white screening, and sequencing. bit.ly/cloningcheck Here’s the original SLIC paper: Li, M.Z., Elledge, S.J. (2012). SLIC: A Method for Sequence- and Ligation-Independent Cloning. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press. doi.org/10.1007/978-1-61779-564-0_5 Here’s a nice blog post from Addgene on SLIC: Plasmids 101: Sequence and Ligation Independent Cloning (SLIC), By Mary Gearing, 2015 blog.addgene.org/plasmids-101-sequence-and-ligation-independent-cloning Here’s a comparison of SLIC, Gibson, and a few other methods: J5 manual, The SLIC, Gibson, CPEC, and SLiCE assembly methods (and GeneArt® Seamless, In-Fusion® Cloning) j5.jbei.org/j5manual/pages/22.html more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
@GordonChao0508
4 ай бұрын
Does Restriction clone also Homologous Recombination? Why does it need DNA ligase while LIC don't need?
@thebumblingbiochemist
4 ай бұрын
It needs ligase because it is not homologous recombination - no matching pieces
@chuchu7248
Жыл бұрын
AMAZING way to explain!!!!
@thebumblingbiochemist
Жыл бұрын
thank you!
@cies_do
Жыл бұрын
Thank you for this very informative video! ❤
@thebumblingbiochemist
Жыл бұрын
Glad you found it helpful!
@aryarezayi8622
8 ай бұрын
Thank you so much 💓
@matejatadei5671
3 ай бұрын
Isn't this IVA cloning? What are the differences?
@thebumblingbiochemist
3 ай бұрын
I hadn't actually heard of IVA, but looking it up, that appears it does multiple PCRs together rather than separate amplifications
@ezez2116
Жыл бұрын
Hi I have one quesiton from T4 reaction part in this video. When I mix vector and insert PCR product to form plasmid what I want, should I have to add other buffer or dH2O? In other words, just adding insert and vector PCR products can make plasmid what I want?
@thebumblingbiochemist
Жыл бұрын
Normally, I just dilute in water
@ezez2116
Жыл бұрын
@@thebumblingbiochemist so basically the plasmid will be made with just adding insert and vector after T4 polymerase reaction. Did i understand correctly...?
@thebumblingbiochemist
Жыл бұрын
The bacteria will do the hard work :) It doesn't actually get stitched together until it's in there
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