Gene Cloning (LIVE DEMO)
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- Опубліковано 29 вер 2024
- Gene cloning is the process in which a gene of interest is located and copied (cloned) out of all the DNA extracted from an organism.
In standard molecular cloning experiments, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into host organism, (6) Selection of organisms containing vector sequences & (7) Screening for clones with desired DNA inserts and biological properties.
Amazing that such a procedure that is a result of incredibly advanced scientific research by the scientific community, can just be carried out on a desk with a set of instruments that do not look too advanced at first glance... Thanks for showing !
so my takeaway from this is ...
Gene cloning involves the use of a cloning vector, such as pbr322, and the gene of interest.
Both the vector and the gene are cut using restriction enzymes, such as EcoRI, leaving flanking ends for ligation.
The ligation is performed in the presence of ligase at 16°C overnight.
The host for the gene cloning is E. coli, specifically the DH5 alpha strain.
On the first day, the host is revived and inoculated in Luria broth for overnight incubation at 37°C.
The following day, competent cells are prepared and transformed using the recombinant DNA from the ligation.
After transformation, the colonies are selected based on their fluorescence under UV light.
Non-fluorescent colonies are recombinant, while glowing colonies are non-recombinant and have intact GFP genes.
This is divided into three days:
the first day for reviving the host and setting up the ligation,
the second day for preparing competent cells and transformation,
and the third day for colony selection.
Bullet Points:
- Gene cloning involves a cloning vector and a gene of interest
- Both are cut using restriction enzymes and ligated in the presence of ligase
- E. coli DH5 alpha is used as the host
- The practical is divided into three days: host revival and ligation, competent cell preparation and transformation, and colony selection
- Non-fluorescent colonies are recombinant, while glowing colonies are non-recombinant and have intact GFP genes.
So far so good?
very detailed explanation.Thank you very much
Thank you sir 🙏
Always İndians! Its always İndians who helps us in any subject! Bless you!!! 🤗
Thank you..
Thankyou for the detailed explanation.
Sir.. it's amazing to see a live demo of molecular cloning.. please make more videos on molecular cloning of proteins Sir🙏😊
Thank you
Nice effort.
Well explained, thank you
We want more videos on Biotechnology sir ❤️
Sure... I will try to make more videos...
Highly informative sir..Thank you 🙏
sir it is really helpful..but want to ask you that the 4th step of the protocol which is incubate at 37C at 300 rpm had been missing in the video.... Please help me regarding this.
Well explained! Thank you sir for this awesome video
Really very informative... Thank u sir... Great efforts
Thank you so much Sir...i am grateful for your help
Step by step explanation with ease. Thank You very much Sir.
If we have any personal queries or any sops we require. Can we ask your assistance?
So nicely elaborated and performed smoothly...🙏🏻🙏🏻
THANK YOU SO MUCH 🙏🙏🙏🙏
Sir make practical video on Elisa
Awesome Explanation sir ❤️
very help full video for my end sem practical thank you sir you r a "w person💪 "
It's a very amazing and informative video for an lab unexperienced students. Thanku for your efforts.
Sir, I Truly admire your explanation and it was so clear. But, I could not resist commenting the fact that the personnel protection was completely off here. As a teacher, you must encourage wearing gloves while handling microorganisms. This experiment could have gone wrong in so many ways for instance, you were opening vials using the hand which the pipette due to which your tip was swinging in air and touching the surface thus bringing in contamination of nucleases. Also, its a fire hazard to use a burner inside a laminar hood/biological safety cabinet.
Please don't take this wrong, I just mentioned all this because young students curiously watch such videos and end up getting the wrong idea about personal protection while working in the lab. It's a request that you kindly use proper gloves and try opening vials with your left hand as you hold the pipette in right. Thanks! Great explanation :)
Yeah you are true... Actually this video was made during COVID period for students and that time there was huge scarcity of personal protective equipments!!!!!
Anyway thanks for your comments...
@@NirajSingh1979 thank you for the response. Yes, covid situation explains a lot.
I hope to see more videos from you 😊have a nice day!
Best video of cloning and transformation on UA-cam
In P plate, you said that only the plasmid has been put for the cell to get transformed. Your plasmid had GFP as well as antiobiotic resistance gene, the how will you explain about those colonies in P plate which are not flourescent and yet resistant to the antibiotic? If they have antibiotic resistivity, the why they don't have the fluorescence?
Hello sir. after ligation u incubate the reaction for 24 h but in our case we are using pcr clone jet kit and it's protocol suggest to incubate only 30 minutes
thankyou sir for helping me with my final year project.
Great work Dr singh
Lecture well understood
Thank you sir..it was very informative
Sir ARMS pcr aur nested pcr ka bhi video bana dy.
Thank you so much for making this video sir!
Thank you for the excellent experiment!
Hello sir....I want to know if all the procedures need to be done in sterile condition after the 3 steps you mentioned for viable cells..? how to maintain that condition? Or not needed?
You have not taken od before the centrifugation step...
Very nice video. Thank you. Please use gloves. For safety.
Thanks for your suggestions. These videos are made during Covid crisis when gloves were not available in the market
sir.. can we use colony pcr to check transformed bacterias?
Yes, ofcourse we can...
Thankyou ...it takes a great effort to make a video.
Keep up the good work.
Understood really welll
Thank you
Awesome
Excellent Sir, thank you for sharing this full procedure.
Thank you
Thank you sir
sir can we this plasmid dna as an standard in real time pcr?
Yes
Sir is it non kit method of cloning?
Yeah, in some portions kit materials has been used..... It is combination of kit and non kit...