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!
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"
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.
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!
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.
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.
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.
@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"
Yes, you can see in this animation that BamHI sticky end on the plasmid matches BamHI sticky end on the insert. Same goes for the other side of the insert cut with NcoI
@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.
i have a question, once you finish the DNA ligation, as the restriction enzyme is still present in the solution, how do you make sure if will not cut the plasmid again? Thanks
I don't know if you still need the answer, but after you perform a restriction digest with restriction enzymes, you can do a heat inactivation step at high temp (usually 65 degrees C, but depends on specific enzyme) for about 20 minutes to denature the enzyme so it loses its activity before attempting to ligate. Other times, the digested DNA is purified away from the restriction enzymes before doing any ligation step.
What if you want a smaller piece of the DNA fragment that is already ligated into the vector? Like, not all the DNA fragment, just 400bp from it, for example.
Hey tk, I did not really get your question. It seems like you want to cut out a fragment from an existing plasmid and clone it into another vector. Is that correct?
Yes, isolate that particular fragment and sequence it. I could've use the steps you instructed in this video but it's already ligated into a vector. So I have to start from there.
Andriy Nemirov Yes, isolate that particular fragment and sequence it. I could've use the steps you instructed in this video but it's already ligated into a vector. So I have to start from there.
Hey! Thanks for this great video! I have a question about the relation of insert:vector -> what could possible go wrong if someone added way too much insert? Would this be of a problem?
Is treatment with alkaline phosphatase necessary for cloning? I know it removes the 5' phosphates so that the vector does not recircularize during the ligation reaction, but I do not think it was covered in this video. Will treatment with the phosphatase allow for more inserts before the transformation process?
The phosphatase is used when vector is linearized with only one restrictase, for example BamHI only. You can still clone into it, but most of your clones will contain your original plasmid that closed onto itself without taking in the insert. But, if the ends have been treated with phosphatase, then self ligation will not happen
Aren't the initially cleaved short pieces of from the cleavage of the DNA and cloning vector able to ligate to the vector and DNA...hence preventing the sticky ends from joining the gene to the opened vector?
Hey I know you asked this a month ago but I thought I'd chime in. Normally we add an enzyme called alkaline phosphatase directly to the vector digestion which removes necessary phosphate groups (from blunt or sticky ends) for ligation. So, the only way you can have a successful ligation is if the phosphorylated insert is ligated to the dephosphorylated vector. hope that helps :)
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.
![ILLSLICK - KILLSHOT REMIX [FULL VERSION]](http://i.ytimg.com/vi/RIK8RrqIAzE/mqdefault.jpg)
I actually like how this video shows you transfers between all of the vials. It makes it easier to follow.
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!
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 =)
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"
Very clear and elegant video, simply beautiful. Love that you posted the script as well. Bravo. Thank you for this.
Thanks!
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.
awesome, very, very, very, nice. Simply explain but full of content
Your words are very simple but most powerful . It's amazing .I didn't know the clonning concept wasso simple .
this helped sooo much!! I had no idea what my textbook was saying lol... awesome video!!!
Very short and full explained in a clear way, Thankyou so much.
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!
A very good and clear demonstration. Thanks
Thank you SO MUCH for this video! Ten times better than my explanation in class! Im gonna ace my exam now!
This is brilliant!!! So concise and easy to follow!
Thanku sir for this brilliant animated vedio this helps me a lot
Actually it's amazingly brief, good job 👍
Very clear and basic demonstration.
Thank you so much for a great and brief explanation. It's definitely a life-saving video.
Very good video quality and detail, although some parts are longer than they need to be because they wanted to show cool graphics.
You are literally a life saver!! Thank you for explaning so well
Hey, thanks!
The sound track is called Tech Talk, it is by Kevin McLeod. I have put the link in video description.
Thanks for the Video, It's simple but very useful.
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.
So good and clear explanation. Thank you.
Very illustrative! Thank you! :)
До боли знакомый голос :))
Seriously: Every bio, biochem etc. freshman MUST see this video!
Great refresher video!
BGM is selected very nicely
Thanks lot sir really you explain very nice 🙏🙏🙏🙏
thanks fr the video it helped me fr my exam preparation
funky music you got there
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.
what happened at 6:14? The bacteria died and released the plasmid?
thank you so much for the easy explanation
Extremely useful, thankyou.
Спасибо за видео, просто и понятно, лучше чем у нас на генной инженерии.
Спасибо за просмотр
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.
great video, easy to understand. thanks a lot
Amen!💪
Thank you sooooooo much.
GOD bless, this video is leaving me questionless
the music is dope... i cannot find it on the link though... give its name please...
The soundtrack is called "Tech Talk" author Kevin McLeod. Google it
Andriy Nemirov thankyou 😁
@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"
Very good video!
This is such a helpful video!THANK YOU SO MUCH!
Thank You Buddy. U made Molecular Cloning Interesting
Super video. Thanks for that.
it was very useful and incredibily easy to understand :D you're great, thank you! :P
Wonderfuull animation,
Thank you sir
I love biotechnology , I love molecular biology , I love my job...
Plz make s similar video on molecular markers .....will really help ✌thanku
its very much helpful......
I like so much this video, it's a great job, congratulations!! greetings from Mexico.
Incredible tutorial!
Thank you so much. I can now understand this better.
Very helpful to visualize what's going on! Thanks for sharing with us.
And I liked the music too :)
great job,really! i did't understand that before i saw this vid XD
Very helpful video.
Love the music.
This is great!! Thank you for sharing!!
Great video! Really straight forward :)
Thank you, thank you, thank you!! I finally understood it clearly!
very helpful!!
You explained very well. Please make some videos on site- directed mutagenesis.
Thank you.
this is a very clear video that visually explains the key steps of molecular cloning. nice
Wonderful video, thanks
How can I check my ligation have no problem or no handling error ?
Aren't you supposed to use the same restriction enzyme in order for the sticky ends of the plasmids and the DNA's overhanging nucleotides to match?
Yes, you can see in this animation that BamHI sticky end on the plasmid matches BamHI sticky end on the insert. Same goes for the other side of the insert cut with NcoI
this video was soooooo helpful !!!
It is a very nice animation .
Very well-made.
Great video, thank you!
Thank you very much for this video! Well explained! You helped me a great deal! ;)
It helped me a lot, thank you very much.
@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.
Hey Andiry :)
we wathced this video today in school- it was really cool Thank You :)
But can u give me a link for the music? TY
Thanks a million! I really mean it
i have a question, once you finish the DNA ligation, as the restriction enzyme is still present in the solution, how do you make sure if will not cut the plasmid again? Thanks
I don't know if you still need the answer, but after you perform a restriction digest with restriction enzymes, you can do a heat inactivation step at high temp (usually 65 degrees C, but depends on specific enzyme) for about 20 minutes to denature the enzyme so it loses its activity before attempting to ligate. Other times, the digested DNA is purified away from the restriction enzymes before doing any ligation step.
What if you want a smaller piece of the DNA fragment that is already ligated into the vector? Like, not all the DNA fragment, just 400bp from it, for example.
Hey tk, I did not really get your question. It seems like you want to cut out a fragment from an existing plasmid and clone it into another vector. Is that correct?
Yes, isolate that particular fragment and sequence it. I could've use the steps you instructed in this video but it's already ligated into a vector. So I have to start from there.
Andriy Nemirov
Yes, isolate that particular fragment and sequence it. I could've use the steps you instructed in this video but it's already ligated into a vector. So I have to start from there.
Thanks man really appreciate it love knowledge.
and another question is, is any procedure to check if the fragment is truly inserted into plasmid?
i like it avery much. very descriptive.
Hey! Thanks for this great video!
I have a question about the relation of insert:vector -> what could possible go wrong if someone added way too much insert? Would this be of a problem?
Is treatment with alkaline phosphatase necessary for cloning? I know it removes the 5' phosphates so that the vector does not recircularize during the ligation reaction, but I do not think it was covered in this video. Will treatment with the phosphatase allow for more inserts before the transformation process?
The phosphatase is used when vector is linearized with only one restrictase, for example BamHI only. You can still clone into it, but most of your clones will contain your original plasmid that closed onto itself without taking in the insert. But, if the ends have been treated with phosphatase, then self ligation will not happen
Very good
Nice video...thanks
very interesting video thanks
Aren't the initially cleaved short pieces of from the cleavage of the DNA and cloning vector able to ligate to the vector and DNA...hence preventing the sticky ends from joining the gene to the opened vector?
Good point. They are actually removed from test tube in a separate clean up step that is not in this animation
Hey I know you asked this a month ago but I thought I'd chime in. Normally we add an enzyme called alkaline phosphatase directly to the vector digestion which removes necessary phosphate groups (from blunt or sticky ends) for ligation. So, the only way you can have a successful ligation is if the phosphorylated insert is ligated to the dephosphorylated vector.
hope that helps :)
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?
helpful... good.. thank you very much
muchas gracias por este interesante y didáctico video.
What software you used in making this video
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.
would they not have cloned if the insert had not been added in?
is there any video on expession DNA cloning?? please need help with it
@CloningStrategies why are two different REs used?
I'm doing a research about cloning and there are two types, molecular cloning and organism cloning I'm still kinda confuse about it.
Thanks sir
Perfect thank you so much
Why did the DNA need to be added to a plasmid and an organism, if only copies of DNA were required couldn't we have just photocopied them in PCR?
Exactly, my question too
is this process still used in 2021?
Ohh yeahh... THANK YOU- this track is so cool for chillin or something :) i luv u :3