TOPO Cloning - TOPO-Blunt, TOPO-TA, TOPO-directional

[@bappadityachandra10]

Please keep making such interesting videos.

[@НикитаСамохинпервыепятьлет]

Thanks you from Russia for the unic content! ❤

[@X.N.M]

Thank you Sir for putting so much effort in making such a great video lecture😢❤❤❤

[@mattharvey8712]

Bravo.........do one on insulin made from yeast.....cheers

[@bappadityachandra10]

Thank you so much for this detailed explanations. It helps a lot to understand the process clearly.

[@zhifeiluo7536]

Incredible explaination. Smart technology!

[@somakhaled5487]

Thank you very much ♥️♥️♥️♥️♥️♥️♥️♥️♥️♥️

[@thanvikanumilli6527]

Your videos are amazing and they are very helpful for me I really love the videos and the content in the videos

[@curlygirl5925]

great content!

[@ciwslc]

Hi, thank you for your video, amazing explanation. I am curious about the screening methods that can be used to detect right orientation of the insert in the case of Topo blunt cloning. Is it done after transfection? what is the process?

[@theCrux]

Do you mean Transformation (not transfection)? In that case, yes. You transform bacteria with the TOPO reaction, get colonies, get plasmids from a bunch of them and then screen for orientation. There are many ways to do so: Restriction digest, Sanger, PCR etc. This video on plasmids goes into screening methods: kzbin.info/www/bejne/aKGkfmB8iat8aMU

[@ciwslc]

@@theCrux Thank you! I saw the video, another incredible one! So, for screening for right direction, Colony PCR follow by Sanger sequencing are a good approach?

[@theCrux]

Yes, that seems reasonable (although most people would just stop after their colony PCR shows appropriate results; and Sanger only if they have a CDS and they care about ORFs/mutations etc.). More commonly, you would extract plasmid from a bunch of colonies (mini-prep) and then directly Sanger on the plasmid and/or do restriction digest (with this approach, colony PCR is not required). Colony PCR (followed by Sanger, if necessary) is a quicker alternative if you are screening for "many" colonies and therefore don't want to spend time/resources on mini-preps.

[@aranyamandal4904]

In sticky topo ta cloning how the insert will have 5' dephosphorylated nucleotide , otherwise the topoisomerase wouldn't be replaced by template nucleotide of 5 prime On 3 prime you told the A overhang and vector nucleotide can be ligated after into the bacterial cell .

[@toonishwarhead]

PCR primers typically come as de-phosphorylated. Please re-visit my commentary on PCR primers at around 6:10