First of all congratulation for your advanced teaching skills. I wanted to ask you a specific question, you said that restriction enzymes are used to obtain separated genes. Now the question, how can, a restriction enzyme discriminate exactly the beginning and the end of a gene? A restriction enzyme cuts the DNA in specific locations in which a characteristic nucleotide arrangements (for example EcoRI cuts G/AATTC), therefore these enzyme could also cut in the middle of gene sequences, this will only produce gene fragments and not entire genes.
The restriction enzyme is selected so that the DNA fragment has just one restriction site (so the "characteristic nucleotide arrangements" is repeated only once).
As we already have knowledge of Nucleotide sequence of genes and recognition sequence of restriction enzymes we can select appropriate enzymes which doesn't cut through the genes(as per my knowledge)
If the restriction enzyme has specific site to cut DNA, how it cuts desired region of human DNA? for example the insulin gene. Is there the palindromic sequence at the edges of desired human gene?
How do you individually separate the four different transformed cells in step 6? Wouldn't they look the same and be all mixed together on an agar plate?
I think they use radioactive labelled probes that hybridised with the desired sequence and then that bacteria containing that plasmid will be multiplied to produce libraries containing that single gene!
First of all, this video is between many others published by this channel an incomparably great help to conquer my upcoming exams, thank you so much. But I do have one remaining question. At step 5 (~ 6:53) the bacterial cells containing the different plasmids that have grown on the nutrient agar are separately grown to produce a colony of plasmids, BUT how are they differentiated from each other on the petri dish? How do you know which library is being reproduced? Any help is appreciated!
I have actually found a logical answer in my lecture script (for those who are interested). Each of the four individual plasmids can be marked with a specific antibiotic resistance different from the others, for example the first one has an ampicillin resistance, the second one a tetracycline resistance etc. Now you can differentiate using selective media containing the separate antibiotics (multiple plates, good luck not needing to waste too many on double results lol)
Amazing! I just Love how he Draws to help visualize how it works out. I wish all instructors did this!, my instructor just talks and talks and never draws out anything so It is very hard for me to visualize how it works out. I love visuals like this! Thank you AK LECTURES! :)
Well I just take a screenshot when he is on a left side of the screen, wait for him to walk to the right side, take another screenshot and put them into one image xD Works for me
thank you a lot) But i have 2 questions What is a difference between Genetic Library and Genomic library? And why we can not use cloning vectorr instead PCR?
because PCR has two limitations.1st is, In PCR to copy a gene, the gene must be known.For unkown genes we cant make a primer in the PCR process. 2nd is, there is a limit to the length of DNA sequence that can b copied by PCR.
Hi, I really liked the video, but there is something I did not understand. After the transformation, when you plant out your E.Coli cells in the petri dish, do you consider all the colonies that grow up in it to build you genomic libraries or just the colonies that show to have both the plasmide and the insert (recombined vector, not just the vector)?
+Diego Otero (student here, so believe me only if you want to :P + i'm French so excuse my English please ) You could, but PCR makes more mistakes so in the end you don't actually have that many exact copy (the longest the fragment the more errors) ... you then have to sequence the clones to be sure you have actual clones...
because PCR has two limitations.1st is, In PCR to copy a gene, the gene must be known.For unkown genes we cant make a primer in the PCR process. 2nd is, there is a limit to the length of DNA sequence that can b copied by PCR.
Saving my life. I like to read my textbooks but sometimes you gotta just hit 1.75x speed and bang out 15 AKvideos
Haha true
On allah
You are the reason I will do well on the MCAT. Thanks.
I NEED an update.
How did you do bro ?
Where are you , did you succeed .....
So is cDNA library and gene library same?
Sir I really like the way you speaking thank you so much.
First of all congratulation for your advanced teaching skills.
I wanted to ask you a specific question, you said that restriction enzymes are used to obtain separated genes. Now the question, how can, a restriction enzyme discriminate exactly the beginning and the end of a gene? A restriction enzyme cuts the DNA in specific locations in which a characteristic nucleotide arrangements (for example EcoRI cuts G/AATTC), therefore these enzyme could also cut in the middle of gene sequences, this will only produce gene fragments and not entire genes.
My guess is that this does occur but only in a small amount relative to where we want the enzyme to cut.
The restriction enzyme is selected so that the DNA fragment has just one restriction site (so the "characteristic nucleotide arrangements" is repeated only once).
As we already have knowledge of Nucleotide sequence of genes and recognition sequence of restriction enzymes we can select appropriate enzymes which doesn't cut through the genes(as per my knowledge)
THANK YOU, your videos are so dense and detailed.
Your explanations are clear and specific. Thank you so much
If the restriction enzyme has specific site to cut DNA, how it cuts desired region of human DNA? for example the insulin gene. Is there the palindromic sequence at the edges of desired human gene?
How do you individually separate the four different transformed cells in step 6? Wouldn't they look the same and be all mixed together on an agar plate?
I had the same doubt
I think they use radioactive labelled probes that hybridised with the desired sequence and then that bacteria containing that plasmid will be multiplied to produce libraries containing that single gene!
+1
clone picking
I know it is a year later, but he kind of explains this in the next video. They use gel electrophoresis to separate by length.
You are lit 🔥🔥 sir.. love from budgam kashmir
First of all, this video is between many others published by this channel an incomparably great help to conquer my upcoming exams, thank you so much.
But I do have one remaining question. At step 5 (~ 6:53) the bacterial cells containing the different plasmids that have grown on the nutrient agar are separately grown to produce a colony of plasmids, BUT how are they differentiated from each other on the petri dish? How do you know which library is being reproduced? Any help is appreciated!
I have actually found a logical answer in my lecture script (for those who are interested). Each of the four individual plasmids can be marked with a specific antibiotic resistance different from the others, for example the first one has an ampicillin resistance, the second one a tetracycline resistance etc. Now you can differentiate using selective media containing the separate antibiotics (multiple plates, good luck not needing to waste too many on double results lol)
Amazing! I just Love how he Draws to help visualize how it works out. I wish all instructors did this!, my instructor just talks and talks and never draws out anything so It is very hard for me to visualize how it works out. I love visuals like this! Thank you AK LECTURES! :)
Hey, you're very talented in pedagogics, so many thanks! :D
Superb explanation
Thank you very much
Is there a way that we can save a picture of the dry erase board behind him? That would be really helpful when reviewing his notes
Well I just take a screenshot when he is on a left side of the screen, wait for him to walk to the right side, take another screenshot and put them into one image xD Works for me
@@magdabielecka8374 same haha
Thank you.
Such a detailed lecture sir.
Can not the gene library be created by using PCR? It will be more time consuming. Need suggestions
sir plz upoad a vedio on maxam gilber method of dna sequencing
You are just a different type of human being. You are above a genius. You are a spirit. I love you.
i was following him until he said that the plasmids broke down the restriction enzymes
Love from India 2022
so is lt like doing PCR in a bacteria instead of a machine?
now i finally get why it is done... thank you!!!
Thanku so much sir.
SIR WHAT DO YOU NOT KNOW
thank you a lot) But i have 2 questions What is a difference between Genetic Library and Genomic library? And why we can not use cloning vectorr instead PCR?
because PCR has two limitations.1st is, In PCR to copy a gene, the gene must be known.For unkown genes we cant make a primer in the PCR process. 2nd is, there is a limit to the length of DNA sequence that can b copied by PCR.
Genetic library further divides into two: viz, Genomic library and cDNA library
THIS IS VERY HELPFUL
Please make a video lecture on Mitrochondrial biogenesis
I love genitics
Hw every gene know their plasmid to stick on it?!
Hi, I really liked the video, but there is something I did not understand. After the transformation, when you plant out your E.Coli cells in the petri dish, do you consider all the colonies that grow up in it to build you genomic libraries or just the colonies that show to have both the plasmide and the insert (recombined vector, not just the vector)?
my best teacher .i wanted to met him sir
Exam time Savior!
You are a life saver. Thanks
One of the great teachers.........i've experienced......!!!
Thank you! Very well explained
Explained very effectively.Thankx a lot
Thank you so much for the explanation
very good presentation and patience....
Thanks for the detailed explanation about gene library.
great explanation ..u make every thing simple,thx alot😊
Very informative and clear explanation! Thanx!
I love u! you saved me!
I love your lectures
Thank You!! Just Saved Me Again
i am from pakistan
Can't we use PCR to amplify each gene instead of using plasmids?
+Diego Otero (student here, so believe me only if you want to :P + i'm French so excuse my English please ) You could, but PCR makes more mistakes so in the end you don't actually have that many exact copy (the longest the fragment the more errors) ... you then have to sequence the clones to be sure you have actual clones...
i think if we use bacterial plasmids to amplify those genes we also would get the proteins cz we use a living cells instead of pcr?
because PCR has two limitations.1st is, In PCR to copy a gene, the gene must be known.For unkown genes we cant make a primer in the PCR process. 2nd is, there is a limit to the length of DNA sequence that can b copied by PCR.
It's way easier to grow bacteria with plasmids rather than to perform PCR. Especially if the DNA is already in a plasmid.
PCR has a limitation like the size of the gene.
and what happens to the bacteria that employ plasmids are non-recombinant?
those are blue..because there beta galactosidase is still functioning...the ones that are transformed and recombinant turn white
how we can get your lecture sir
Un ma-es-tro.
Outstanding.
your awesome dude thanks
Great explanation
very good
Thank you! :)
Wow!
thank you man! You save all my year
Carlos Vasconcelos you're welcome! :)
This made the impossible, possible for me. Thank you so much. :)