girl thank you so much for making this video! this is so good quality and free 🥺 thanks for giving me some core info to be able to start my assignment 💛💛💛
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.
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 ?
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.❤
thank you so much for this incredibly helpful video! i'm currently an undergrad and was so confused when my PI explained it to me. I clicked on your channel and to my surprise, there are hundreds of other videos like this one. I feel like I struck a goldmine haha. I appreciate you!! 🫶🫶
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?
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?
I'm a new master student about molecular biology, your videos 're helpful. Thank you so much. Wish you all the best!
I'm interning at Biochemistry lab and know very little! Your videos help me play catch up! Thank you!!
Happy I could help! Good luck with the internship!
girl thank you so much for making this video! this is so good quality and free 🥺 thanks for giving me some core info to be able to start my assignment 💛💛💛
happy I could help! Good luck!
i have a biochem exam coming up in two days, and this video was really helpful. Wish me luck!😊
Hope it went well!
How great the video is. It made me more familiar with cloning knowledge!
Glad it was helpful!
Awesome finding your videos. Will watch all of them.
Glad you like them!
Hi! This is so informative and so easy to follow-and exactly what I needed- thank you!
That's great to hear - thank you! So happy I could help
Wow! You are so good! I wish you a great career in science!
Thank you!
Thank you so much for this content! It was really helpful 💙💙💙
So great to hear!
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.
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.
This was so helpful! I loved your infographics
I'm so glad!
thank you so much for this incredibly helpful video! i'm currently an undergrad and was so confused when my PI explained it to me. I clicked on your channel and to my surprise, there are hundreds of other videos like this one. I feel like I struck a goldmine haha.
I appreciate you!! 🫶🫶
Thank you so much! I'm sincerely super happy to know you found this (and others) helpful. Best of luck with your studies!
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
Great video and explanation! 😃
Thank you!
Excellent video thanks!
Thanks!
Thanks you my sister from yemen
So glad I could help!
good job💯
Thanks!
So nice 😂