Plasmids and Recombinant DNA Technology

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[@dayhdez]

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[@AKLECTURES]

+dayhdez Thank you for those kind words! greatly appreciate that :)

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I learnt more in these 14 minutes than 2 weeks of lessons, wow thank you

[@AKLECTURES]

+Simona Peneva you're welcome! :)

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Really clear explanations. Constant repetitions makes it really easy to follow and to retain the information. Love this teaching method, keep it up! Was able to watch at 2.3x and the pronunciation was still crystal clear!

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[@bmrasta5943]

Great source of information! Thanks for taking the time to spread education on recombinant DNA technologies.

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by far the best video on this material!

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Even though I am only in the 4. min. of the video, you explain it in a much more understandable and fluent way than my professor. thank you so much

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[@MrRoperete]

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[@GeAsita]

Absolutely clear video of recombinant DNA. Congratulations and thanks a lot for sharing, keep it up!!!!!

[@AKLECTURES]

Gabriel Amodeo Thanks Gabriel! I will :)

[@asanda6315]

This was so helpful..your explanations are so clear, Thank you!

[@laurilintott4754]

Maybe you cover this in a future lecture but you have glossed over the main difference between pBR322 and pUC18, and that is how you determine which cells have taken up a recombinant plasmid (as apposed to taking up just the starting plasmid). In pBR322 you must use replica plating to determine which cells have taken up recombinant plasmid vs which cells have taken up the starting plasmid. For example, if you were cloning into the tetracycline gene, you would first select with ampicillin in order to identify which cells took up the pBR322 plasmid. You would then check the ampicillin colonies to determine which ones were no longer tetracycline resistant. Ampicillin resistant and tetracycline sensitive cells will have the recombinant plasmid. If the cells are resistant to both antibiotics, then they took up non recombinant pBR322. With pUC18 you can tell in a single step which colonies took up recombinant plasmid because these cells will be ampicillin resistant but will not turn blue because the inserted sequence has disrupted the B-gal. pUC18 has several other advantages over pBR322 but the ability to screen for recombinant plasmid in one step instead of two is a major advantage.

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Great explanations, much appreciated.

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Very grateful I found this video! Crazy how much I pay in tuition at my college and yet I learned much more from this video than from my professor.

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Great explanation, Great teacher skills!! This video was very useful to me. Thanks!!

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[@AKLECTURES]

+Dyane Ribbink you're welcome Dyane

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[@AKLECTURES]

you're welcome Sarah! best of luck with your studies!

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[@user-ph1xh9bj6w]

Such a detailed explanation! At "Recombinant DNA: Genes and Genomes - A Short Course" book about the last section of your video says that the plasmid does not contain the whole beta-galactosidase gene back the part of the gene that produces the enzymes N-terminus (part a). We also have bacteria that contain the part of the gene that produces the other part of the enzyme (part Ω). When a and Ω are together they produce the active form of the enzyme. However, I do not understant why do we make our lives more complicated, but it seems that the first part of the gene is used for the production of a lot of biopharmaceutical products, and my question is why do we use only a part of beta-galactosidase gene and not the whole one? Is it because of the size limits?! Keep spreading your endless knowledge!

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[@rachetysambaburajendrapras2715]

Very nicely explained about the r DNA technology.

[@mominjaved7616]

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Very helpful! Thanks!

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[@yosigilad481]

Wonderful lecture! There is a small mistake I indicated at the end of the lecture (where the inactivating incorporation in the beta galactosidase site is explained). The solution will not turn blue in any case (either the cells successfully uptake the plasmid or not), because the beta galactosidase is inactivated. The only indication will be is if the recombinant DNA was successfully incorporated (no blue color then) or not (then the solution will turn blue).

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Thank you so much! Please come teach my bio class!

[@calebm9000]

Wouldn't it be more efficient to add ampicilin to the solution since that's the gene you inactivated, or do the bacterial plasmids not have resistance for either one?

[@egbejustine]

Does it mean that prior to screening...the host cell (bacterium) that is to be transformed would have it's plasmid rid off...?? Saying this because we know that bacterium cell has plasmid in it. There might be a possibility for a plasmid that is in a bacterium cell may not take up the recombinant DNA but would confer Antibiotic resistance

[@mohsenteimouri9071]

Hello, You inactivate the ampicillin resistance region. It means if we add ampicillin antibiotic to the colony, the bacteria that take DNA fragments will not survive. However, you mentioned tetracyclin will die the cell. While the tetracyclin regions are intact. Am I right?

[@irenevitali6187]

how can we get the little DNA fragment to locate into the plasmid if the restriction enzymes cut the DNA molecole without create a little fragment? I don't know if I express my doubt in a clear way...

[@veronicacarvajal4138]

What is the significance of inactivating the green gene and having the cell pick up that plasmid, when both the green gene and blue gene were activated to begin with? Wouldnt a cell want a plasmid that enables resistance to both ampicillin and tetracycline?

[@morganmiller1282]

Why do we use more than one antibiotic and sometimes even three of them in our plasmid? Wouldn't we only need to put in one resistance gene to see if our plasmid was taken up in a cell?

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