Key Steps of Molecular Cloning

Molecular cloning is a process of isolation of a specific DNA fragment and transfer of this fragment into a plasmid vector.
As a part of the plasmid vector, the DNA fragment could be easily amplified, sequenced, stored for long periods of time, and used for gene expression and other functional studies.
Two starting ingredients of molecular cloning are a plasmid vector and a DNA fragment that has to be inserted in it.
The DNA fragment is usually a gene or other functional region from a living cell, or it could be an artificial sequence with properties useful for a researcher.
Plasmid vector is circular piece of DNA that could be easily amplified in E. coli, stored for long periods of time, and easily manipulated in a test tube.
A typical plasmid vector contains:
- an origin of replication that allows it to be replicated inside a bacterial cell;
- a selection marker, for example a beta lactamase gene coding for ampicillin resistance;
- and a multiple cloning site , which could be cleaved with several restriction enzymes, such as BamHI (G-GATCC), EcoRI (G-AATTC) or NcoI (C-CATGG).
In many vectors, the multiple cloning site is surrounded by sequences of promoter and terminator, that guide expression of inserted genes after the vector is introduced inside a cell.
To be used for molecular cloning, both vector and insert DNA are treated with restriction enzymes that cleave double stranded DNA molecules producing overhanging single stranded nucleotide tails.
After their ends have been prepared with restriction enzymes, vector and insert are combined together, and another enzyme, called a DNA ligase is added to the mix.
At the same time as complimentary base pairing of single stranded overhands brings the ends of vector and insert together, the DNA ligase fuses them into one intact DNA molecule.
In order to make multiple copies of this molecule, the ligation mixture is introduced inside the E. coli cells in a process called transformation.
During the transformation the cell-DNA mixture is kept on ice and then exposed to 42 oC.
Such sudden change in temperature drives the DNA inside some of the E. coli cells.
Then the cells are plated on a plate with growth medium supplemented with a selective antibiotic.
Only the cells that acquired the plasmid have resistance to the antibiotic and are capable of growth on such a medium.
After overnight incubation at 37 oC each transformed cell produces a colony of identical cells, oftentimes called a clone.
The selected clones are then individually picked, grown even further in a liquid medium, and the DNA is extracted from them.
Thus, in the process of molecular cloning, a DNA fragment that represented a tiny fraction of cell genome is integrated into a bacterial plasmid.
As a part of a plasmid, this DNA fragment represents a quarter or more of total DNA in a test tube and it could be effortlessly and endlessly amplified in E. coli.

[@annabashkirova781]

I actually like how this video shows you transfers between all of the vials. It makes it easier to follow.

[@Lillith9008]

I like the pace in which you did the video, the music and the animation that went with it, it was very clearly laid out and made it easier to understand - thank you!

[@FancyUnicorn]

Very clear and elegant video, simply beautiful. Love that you posted the script as well. Bravo. Thank you for this.

[@CloningStrategies]

Thanks!

[@sargonhani6]

You sir are an amazing instructor, I wish some professors would watch your channel to learn how to lecture. Thank you very much for your videos =)

[@CloningStrategies]

Spot on! After restriction digest the DNA is separated on an agarose gel. Small fragments are removed from the solution in the process, otherwise they would interfere with ligation.
Watch "Simply Cloning - Chapter 4 - Gel Purification"

[@laurenkoval3041]

Namen, (sorry if this is a bit late) but the same restriction enzyme is used in both the vector and the insert. In the video, both BamH1 and Ncol are used in both the vector and the insert cells. These enzymes act on a certain DNA sequence in the cell, causing it to cut the DNA forming sticky ends. You are right. Therefore, where BamH1 cuts the vector cell, and where the Ncol cuts the insert cell (and vice versa), these two areas are complimentary, therefore, causing the cells to fuse with the help of DNA ligase. Sorry if that's a bit wordy.

[@gembarry8280]

awesome, very, very, very, nice. Simply explain but full of content

[@BeautyLovesSam]

this helped sooo much!! I had no idea what my textbook was saying lol... awesome video!!!

[@tmsplltrs]

funky music you got there

[@noorpk]

BGM is selected very nicely

[@sam33n53]

A very good and clear demonstration. Thanks

[@247stoned5]

Thank you.

[@CloningStrategies]

Hey, thanks!
The sound track is called Tech Talk, it is by Kevin McLeod. I have put the link in video description.

[@cansudeniztozkoparan1150]

Thank you so much for a great and brief explanation. It's definitely a life-saving video.

[@shankartradersshankar4811]

Thanku sir for this brilliant animated vedio this helps me a lot

[@taimoorhassan44]

Very clear and basic demonstration.

[@prashantkumarparmanu]

Your words are very simple but most powerful . It's amazing .I didn't know the clonning concept wasso simple .

[@joslypierre-louis1658]

Thank you for posting this video I am working in a biophysical chemistry lab for the first time with no background in biochemistry and was so confused as to what I was doing concerning my internship this video has made it so clear for me THANK YOU!

[@nertoni]

Thanks for the Video, It's simple but very useful.

[@jeffy1218]

This is brilliant!!! So concise and easy to follow!

[@anjaligyawali3720]

Very short and full explained in a clear way, Thankyou so much.

[@freedomtv1505]

Amen!💪
Thank you sooooooo much.
GOD bless, this video is leaving me questionless

[@mohdadilkhan1648]

Actually it's amazingly brief, good job 👍

[@CloningStrategies]

You got everything right, that's pretty much how it is.
By the way, I find that writing a brief summary like this is very got for remembering new information.

[@FONK6969]

Love the music.

[@ramitayadav62]

Thanks lot sir really you explain very nice 🙏🙏🙏🙏

[@sapnarawat8609]

its very much helpful......

[@trilokization]

Very illustrative! Thank you! :)

[@animeloverXinuyasha]

Fantastic video, thank you.

[@TheLiquidFilth]

Extremely useful, thankyou.

[@poetryodia6704]

thank you so much for the easy explanation

[@Christmas7731]

Thank you SO MUCH for this video! Ten times better than my explanation in class! Im gonna ace my exam now!

[@mizuki195]

You are literally a life saver!! Thank you for explaning so well

[@bibifg9375]

great job,really! i did't understand that before i saw this vid XD

[@jessica198929]

very helpful!!

[@fazlehaque2372]

Thanks sir

[@Cheddarific]

Very good video quality and detail, although some parts are longer than they need to be because they wanted to show cool graphics.

[@TarekMG]

Thanks a million! I really mean it

[@indumatisharma3646]

Super video. Thanks for that.

[@silviamuccioli1907]

it was very useful and incredibily easy to understand :D you're great, thank you! :P

[@aghrabattsalem5403]

So good and clear explanation. Thank you.

[@nadhamidx]

Thank you so much. I can now understand this better.

[@EmbryoJenya]

До боли знакомый голос :))
Seriously: Every bio, biochem etc. freshman MUST see this video!

[@CloningStrategies]

@ntlema2 Great questions!
Which enzymes to use: This is what you decide as a scientist when you are planning your cloning experiment. You look at the sequence of the plasmid and see what restrictions sites are available, then you integrate those sites into the primers you use to amplify the DNA fragment. Check "Simply Cloning - Chapter 1 - Planning" video.
To extract cloned DNA: It is more than just a liquid medium. Check "Chapter 8 - Plasmid Miniprep"

[@johnmc2k]

Great refresher video!

[@CM-ef8fu]

Very good video!

[@MohamedKhalidAli]

Thank You Buddy. U made Molecular Cloning Interesting

[@samanthagallagher5909]

Very helpful to visualize what's going on! Thanks for sharing with us.

[@samanthagallagher5909]

And I liked the music too :)

[@yelizidilyigit5716]

great video, easy to understand. thanks a lot

[@CloningStrategies]

@powerzoner If you stop the video at 2:58 min you will see that overhangs from different restriction enzymes don't match each other. So plasmid vector can't close on its own, only with the insert.
There are all kinds of exceptions, but using two REs is a simple way of preventing vector from closing onto itself.

[@slackwatters9604]

Thanks man really appreciate it love knowledge.

[@billowingsatire12]

Incredible tutorial!

[@periebeid6117]

Thank you, thank you, thank you!! I finally understood it clearly!

[@rittenbrake1613]

Very good

[@feb17aaru]

This is such a helpful video!THANK YOU SO MUCH!

[@03500]

Perfect thank you so much

[@CloningStrategies]

Compared to PCR fragments, plasmids are more stable, and they can be amplified inside E. coli at a very low cost.
Also, in order to study gene function, a gene has to be linked to promoters and terminators, which is much easier done in a plasmid.

[@leticiareque5751]

This is great!! Thank you for sharing!!

[@Philisophe94]

Спасибо за видео, просто и понятно, лучше чем у нас на генной инженерии.

[@CloningStrategies]

Спасибо за просмотр

[@arunavnanda7]

thanks fr the video it helped me fr my exam preparation

[@rubi22teresa]

Thanks

[@vanopnt]

i like it avery much. very descriptive.

[@shanupandey8258]

Plz make s similar video on molecular markers .....will really help ✌thanku

[@hannahmeadors6558]

Great video, thank you!

[@Calaf120]

muchas gracias por este interesante y didáctico video.

[@herojit009]

It is a very nice animation .

[@arpitakarandikar8249]

You explained very well. Please make some videos on site- directed mutagenesis.

[@antrisha9868]

THANK YOU !

[@bs0u41dd]

Awesome!

[@Molecular_Machine]

Wonderful video, thanks

[@elmariachirockerocho]

Very well-made.

[@puriashabahng3134]

It helped me a lot, thank you very much.

[@DreamingCimderella]

Why do we have to insert the DNA in the plasmids? Can't we just use PCR, to amplify the fragment of interest and store it?

[@wahyuhidayati244]

Can i dubbed the video with my language-Indonesia? I already used your video for my classes since 2013 by inactivating the speaker (mute). Due to the online class during pandemic, i want to dubb the video. Some of my students do not familiar with english. i love the video and it shows the real recombinant DNA research on the lab.

[@ChommyK]

Very helpful video.

[@kenwatanabe4637]

The main problem with this video is that during the insertion into the plasmid, not all the plasmids will pick up the insert. When the bacteria is heat shocked, some bacteria will pick up plasmid with the insert, some will pick up plasmid without the insert. The video is missing the step to ensure the selected colony picked up the plasmid with the insert.

[@marie2966]

Thank you very much for this video! Well explained! You helped me a great deal! ;)

[@bhimgd]

helpful... good.. thank you very much

[@queseraseraozi]

Wonderfuull animation,
Thank you sir
I love biotechnology , I love molecular biology , I love my job...

[@manannarkar2701]

the music is dope... i cannot find it on the link though... give its name please...

[@CloningStrategies]

The soundtrack is called "Tech Talk" author Kevin McLeod. Google it

[@manannarkar2701]

Andriy Nemirov thankyou 😁

[@CloningStrategies]

Thanks!

[@snehasishmandal9363]

Very very thank you sir

[@youtubyfein]

this video was soooooo helpful !!!

[@nalinirau]

would they not have cloned if the insert had not been added in?

[@coca833]

I like so much this video, it's a great job, congratulations!! greetings from Mexico.

[@alexasmithy]

very interesting video thanks

[@mohammedshannaq3911]

great

[@powerzoner]

@CloningStrategies why are two different REs used?

[@Tomkens1985]

Brilliant! Thanks!!

[@nuzhatkhurshid7318]

Nice video...thanks

[@rinamunalisa4315]

How can I check my ligation have no problem or no handling error ?

[@amralaa1780]

very good

[@ReedCSings]

Great video! Really straight forward :)

[@CloningStrategies]

I've put closed captions ;)

[@NewWesternFront]

what happened at 6:14? The bacteria died and released the plasmid?

[@faizaarshad705]

What software you used in making this video

[@santelmo5222]

Thank you!!!!!!!!!!!!!!!!!

[@shoppittsburghnow]

great vid you have here

[@eckostar61]

is there any video on expession DNA cloning?? please need help with it

[@trif169]

is this process still used in 2021?