Gene Cloning (LIVE DEMO)

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.

[TonyMontana-lmgp]

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?

[myrkwood]

Always İndians! Its always İndians who helps us in any subject! Bless you!!! 🤗

[bailahie]

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 !

[najibyahayasani]

Step by step explanation with ease. Thank You very much Sir.

[awnishkumar]

Best video of cloning and transformation on YouTube

[Zyy]

Sir.. it's amazing to see a live demo of molecular cloning.. please make more videos on molecular cloning of proteins Sir🙏😊

[BPUJAA]

Thankyou for the detailed explanation.

[vivekmanyapu]

So nicely elaborated and performed smoothly...🙏🏻🙏🏻

[ArtistRashmi]

It's a very amazing and informative video for an lab unexperienced students. Thanku for your efforts.

[shobhasurbhaiyya]

Really very informative... Thank u sir... Great efforts

[OtikotimoRonnie]

Great work Dr singh
Lecture well understood

[sangra]

very help full video for my end sem practical thank you sir you r a "w person💪 "

[pranjalkumar]

We want more videos on Biotechnology sir ❤️

[TheLawsoniaLab]

Well explained! Thank you sir for this awesome video

[arindampaul]

Excellent Sir, thank you for sharing this full procedure.

[sadafchaudhary]

Thank you so much Sir...i am grateful for your help

[MIs-kjpx]

thankyou sir for helping me with my final year project.

[tusharkashyap]

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?

[navdeepkaur]

Thankyou ...it takes a great effort to make a video.
Keep up the good work.
Understood really welll

[dhrubasonowal]

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.