Molecular cloning overview - techniques & workflow
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- เผยแพร่เมื่อ 5 ส.ค. 2024
- In MOLECULAR CLONING we take a gene* from one place and (most commonly) stick it into a small circular piece of DNA called a PLASMID VECTOR** → stick that into expression cells to use the gene’s instructions to make that protein → get lots of protein you can purify & study (or study how it works in those cells if that’s your goal). I made a video discussing the basics of the process and techniques that it involves from cloning to transformation to that final sequence check. So check it out (if you want)
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blog form: bit.ly/molecularcloningguide ⠀
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Before you can get that plasmid into the cells you choose, you have to get your gene into that plasmid. The “classic” way to do this is the “cut & paste” with RESTRICTION CLONING. Bacteria have DNA-specific “scissors” called RESTRICTION ENZYMES (aka restriction endonucleases, or REases) that recognize & cut specific “code words” (RESTRICTION SITES aka recognition sequences) written in DNA, which serve as “dotted lines.” With RESTRICTION CLONING, we use these DNA “scissors” to cut an “insert piece” with the gene we want to stick in and a “vector piece” with the vector we want to stick it in with the same pairs of scissors so they have complementary cuts. And then we purify the matching pieces and mix them together, adding a “stitcher” called DNA ligase to seal them up tight. ⠀
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I use a different method, SITE & LIGATION INDEPENDENT CLONING (SLIC). With SLIC cloning we use Polymerase Chain Reaction (PCR) to make lots of copies of (amplify) “insert pieces” & “plasmid pieces.” When we do this copying we add on extra matching bits to the end of the pieces. We then let an enzyme (reaction mediator/speed-upper) chew 1 strand of each of these these ends back a little to leave sticky parts. And then we put them into bacteria to piece them together, fixing the damage. Similar PCR-based methods include Gibson assembly & Golden Gate Assembly. ⠀
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for proteins, a gene is actually kinda like a “pre-recipe” - proteins are actually made from RNA copies of the DNA recipe. These RNA copies are called messenger RNA (mRNA) and they’re edited (and temporary) copies of the gene - editing involves cutting out regulatory regions (introns) and stitching together the “expressed” regions (exons) in a process called RNA splicing. Our cells do this (and can do it in multiple ways to give you alternative splice products (splice isoforms) - kinda like purposefully skipping a step in a triple-decker cake recipe in order to make a double-decker cake). But bacterial cells don’t - and even if they did, they wouldn’t know which splice isoform to make to know what cake to bake!. So, instead of inserting the gene like it occurs in our DNA, we insert a version of the gene that is complementary to the edited form we want - mature mRNA for the specific isoform of interest. We call this complementary DNA (cDNA). To try to avoid confusion, I’ll use the term “insert” to refer to the cDNA we put it (this is actually a more relevant term anyway because you can clone in *any DNA - it doesn’t have to be cDNA unless you want to make protein from it.⠀
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**your vector doesn’t have to be a plasmid and sorry in advance for going back-and-forth between “plasmid” & “vector” - a vector is a “vehicle” for transporting your gene into the cells (analogously to how mosquitos can be vectors for malaria, “driving” the malaria DNA into you when they go to suck your blood). Instead of mosquitos, we use things like plasmids, “artificial chromosomes,” and weakened viruses such as adenoviruses (a kind of cold-causers). What kind of vector you use depends largely on the type of cells or organisms you want to get the DNA into and how much DNA you want to stick in.
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I’m going to focus on molecular cloning of plasmids, because that’s probably the most common (and easiest to visualize and stuff). But one of the beautiful biochemical things about molecular cloning is that you can use the same sort of techniques to stick DNA “anywhere.”
There are numerous options including variations & mixtures of:⠀
🔹restriction-enzyme-based: cut & paste⠀
🔹 PCR-based methods: copy just the parts you want and staple together⠀
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regardless of what method you choose you’ll need 2 things:⠀
1️⃣ plasmid vector you want to stick your gene into (destination) ⠀
2️⃣ something containing the gene or “insert” you want to stick into that vector - these days, this insert is usually already inserted into a different plasmid vector just not the one you want so what you need to do is SUBCLONE it → move it from one plasmid to another instead of “traditional” cloning where you’d be moving it from its original location (such as chromosomes inside human cells)
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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 - วิทยาศาสตร์และเทคโนโลยี
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Thank you so much! I'm sincerely super happy to know you found this (and others) helpful. Best of luck with your studies!
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Thanks!
Nice video👍informative and helpful. I have a question,
I need to clone recombinant protein vector. For that I have ordered primer made from cds sequence of the gene of Interest. These primers have forward and reverse sequence along with that restriction enzyme site. My forward primer also has kozak sequence and flag tag. I prepared cDNA of the gene and put it for pcr with these primers but its not at all amplifying. I even used control cDNA and primers from takaras multiple tissue cDNA kit as positive control and kit control primers with my cDNA for troubleshooting, but no bands came. Can you suggest any method or literature which I can refer?
thanks! sorry you’re having difficulty with your cloning. are you trying to amplify out of cells?
Thank you so much for the great video! I am wondering, when you do PCR cloning and use primers that contain no phosphate groups but your vectors also don't have phosphate groups because you might have used AP (alkaline phosphatase) to remove them because you don't want self-ligation to occur, you use PNK to add phosphate groups. But how do you control adding phosphate groups only to the primers and not to the inserts and not to the vectors when they in the same mixture?
Correction: But how do you control adding phosphate groups only to the inserts and not to the vectors when they in the same mixture?
Thanks! You would do the modifications before mixing them
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So glad I could help!
Great video Brianna 👏, but I still have a confusion~~
After completing SLIC, three plasmid types are present in our samples, right? The template plasmid (produced by bacteria and modified by methylation), the plasmid containing the 'insert' (unmethylated), and the plasmid containing the site-directed mutagenesis.
So my question is: Why did site-directed mutagenesis would happen in SLIC? Is the plasmid containing this mutation a byproduct of PCR in SLIC? As a PCR product if it is not methylated it cannot be degraded by Dpnl, how do we remove it? 🤔
Thanks! You only have a single intact plasmid for each PCR reaction, and then you DpnI it. None of the PCR products should contain the non-mutagenized region.
So nice 😂
Nice video ❤ also The screening technique destroys the cells so isn't it counter-productive? We want to clone the gene of interest and now we destroyed the cells that are going to do so or is there another step after the screening part ?
Thanks! Screening doesn't destroy the cells, so I'm not sure what you're referring to? Can you clarify?
The screening reagent us an antibiotic, typically you would insert a gene coding for antibiotic resistance along with your target gene. Then you are able to select the modified bacteria by using an antibiotic to get rid of the non modified bacteria. Another technique is to add a florescent protein gene and select the colonies that glow.❤
Correct! Using the antibiotics is an example of a selection technique, whereas fluorescence would be screening.