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! :)
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)?
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?
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)
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
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!
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.
+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.
