Erratum: 11:13 - The origin is taken from MB1 plasmid and NOT from ColE1. Note that ColE1 and MB1 are both ori-type plasmids so their "ori" work identically (as explained at 4:36).
20:00 One can use bacterial strain such as DB3.1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of CcdB. Cheers👍
Wait I'm confused how can you tell which bacteria have disrupted plasmids if they are all immune to it's active effects? Or do you mean propagation outside of synthetic lethality? Also dumb questions what does disrupted mean for a plasmid. Also what is a starting plasmid and why is cddb toxic for that even after it is disrupted?
25:53 Isn't it like this that during Alpha complementation, the Lacz alpha only produces 2 subunits of beta galactosidase and the remaining 2 omega subunits comes from the mutated E.coli strain (Delta M15) which also has a defective lacz gene, and due to complementation between these 2 (alpha and omega) makes it a functional beta galactosidase giving blue colour And on the contrast, if our gene of interest disrupts lacz alpha gene, then only omega subunit is produced which is non functional and we get white colonies...
HI, thanks for another brilliant video. I still have one doubt around screening blunt ended inserts derived from Topo Blunt Cloning. Is the Colony PCR and Sanger seq the only way of screening for fragments inserted in the wrong direction?
Restriction digest would be the first thing to try before going for Sanger Seq. Colony PCR works too, but it is prone to false positives given high sensitivity + it needs primers/PCR reaction; so it can get relatively expensive than the other two options.
Erratum: 11:13 - The origin is taken from MB1 plasmid and NOT from ColE1. Note that ColE1 and MB1 are both ori-type plasmids so their "ori" work identically (as explained at 4:36).
20:00 One can use bacterial strain such as DB3.1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of CcdB. Cheers👍
Perfect 🙂
@@theCrux thanks. Your videos are fantastic and keep making more. Best wishes.
Wait I'm confused how can you tell which bacteria have disrupted plasmids if they are all immune to it's active effects? Or do you mean propagation outside of synthetic lethality? Also dumb questions what does disrupted mean for a plasmid. Also what is a starting plasmid and why is cddb toxic for that even after it is disrupted?
Thanks for the lecture 😊
25:53
Isn't it like this that during Alpha complementation, the Lacz alpha only produces 2 subunits of beta galactosidase and the remaining 2 omega subunits comes from the mutated E.coli strain (Delta M15) which also has a defective lacz gene, and due to complementation between these 2 (alpha and omega) makes it a functional beta galactosidase giving blue colour
And on the contrast, if our gene of interest disrupts lacz alpha gene, then only omega subunit is produced which is non functional and we get white colonies...
Yep, those details sound about right.
@@theCrux fairs...
Thank you, your videos helped so much...
Please upload on PCR in detail mechanism and it's different types
HI, thanks for another brilliant video. I still have one doubt around screening blunt ended inserts derived from Topo Blunt Cloning. Is the Colony PCR and Sanger seq the only way of screening for fragments inserted in the wrong direction?
Restriction digest would be the first thing to try before going for Sanger Seq. Colony PCR works too, but it is prone to false positives given high sensitivity + it needs primers/PCR reaction; so it can get relatively expensive than the other two options.
Can you post a video for bacteriophages as vectors both M13 and lambda phage and how we clone our gene of interest in this system
The video for lambda phage is here ruclips.net/video/SHE0lpMiitA/видео.html
17:26 Why can it not replicate?
Because its ori is mutated to RNA pol can't bind properly to it
Hey can you please make more videos like on pET vectors and Bacterial expression system
This series is absolutely 100% helpful especially for Biotechnologists
Keep uploading quality content like these!!! 👍