I found that so many articles/videos discussed the topic of PCR and QPCR in such excess that, as a beginner, I struggled to define the difference in such a way that I could understand it. Your video/s and delivery of information are commendable and I cannot thank you enough for your channel. Superb.
I love when algorithm finally shows a usefull and interesting video after a long time searching for a good explanation. Thanks a lot for your amazing work here.
This video, as well as the videos on Sanger and NGS, are brilliant. This the best molecular biology content I have ever seen on RUclips. Thank you so much, you are so great!!!
*Welcome to ClevaLab* - if you like the video, please give it a 👍 and subscribe so we can reach more people. Also, if you have any questions, feel free to ask in the comments. Get a *PDF Summary* of this video here: www.clevalab.com/post/pcrandqpcr.
This is an excellent PCR overview. I do have one question I was hoping you could answer. When selecting the primers for PCR, some textbooks/websites show the primers binding to the sequences flanking the DNA sequence of interest while others show the primers binding to the very beginning and end of the sequence of interest (not the flanking sequences). Can you explain the reason for this? I thought the primers always needed to bind to the "outside" of the sequence of interest?
Thanks for your question. 🤓 It can depend on the type of PCR you're doing. If you want to clone a specific gene or part of DNA, you will design the primers outside the region of interest. However, if you're doing gene expression, you'll choose a primer within the area of interest, usually across an intron boundary in the mRNA sequence. Primer design might be a good topic for a future video!
@ClevaLab So it basically depends on what application you're using the PCR for. That's kind of what I was thinking, but I wanted to ask anyway. Thank you for your response, and I think it would be great to do a video on primer design in the future!
I have one query.. Primer binds to strand and then it extends but how it's extension stops upto the end of particular gene. How it comes to know this is the end of gene rather continuing extension. Please reply
Thanks for your question. 🤓 For each DNA probe just one dye molecule is present. The most common dye is 6-FAM (Fluorescein), one 6-FAM molecule is added to each probe. To see the chemical structure see this link: en.wikipedia.org/wiki/6-Carboxyfluorescein
So, how do we make these very specific primers, which enable us to 'PCR' one specific gene, if we dont already know the DNA sequence? The sanger method of DNA sequencing, requires specific primers, complementary to the DNA younare trying to sequence. Well hold on a minute, thats a bit of a paradox isnt it. How do you know what the primer should look like if you dont know the sequence of DNA?
Thanks, good question. 🤓 The original method by Sanger to sequence PhiX174 (a bacteriophage), the first full-length sequence made, did not use primers. This was because primers were hard to generate then. Instead, they used restriction enzymes to cut DNA into fragments and used these fragments as the primers for their sequencing reaction. So, they didn't need to know the sequence as they depended on restriction sites to create the first full genome sequence. Restriction enzymes are made by bacteria as a defence against bacteriophage (a virus for bacteria). They cut up the bacteriophage DNA to restrict its ability to reproduce. This fact was used by Sanger in combination with radiolabeled nucleotides. Today, if a sequence is unknown, you can use random hexamers to amply it in a PCR or perform shot-gun or full genome sequencing without knowing any sequence. Short DNA fragments of known sequences are stuck on the ends. These known DNA fragments are then used to sequence the unknown DNA.
thank you a lot; explanation with your animation is excellent and the flow ask for more......seven minutes viewing seems like 2 mins. .. thank your lots of work of animation behind for sharing knowledge🤩
These videos always miss a very important fact that nucleotides are free-floating around with the rest of the slurry, and are snatched up by the DNA-polymerase as they pass by in order to be used for the copying.
Thanks for your comment. I'm so glad it helped you. 🤓 Sure, you can find images in the blog post or even download a pdf. Find it here: www.clevalab.com/post/pcrandqpcr
Hi Abby, thanks for your question; it's a good one. Standard PCR can be semi-quantitative. We used to used this method in the lab before qPCR was available. Suppose you stop the PCR during the exponential growth phase of the samples. Then, you can compare the DNA level between samples. But, the dynamic range is tiny in comparison to qPCR. So these days, people use standard PCR for yes/no type testing. I hope this helps and answers your question. Please let me know if you have any further questions. 🤓
@@ClevaLab Ohh, but in what phase in the amplification determines the "yes" or "no" answer? Is it in the plateau phase? Also, if I were to optimize a PCR reaction, is it okay if I use Agarose Gel to determine the quality of DNA result through crowning? Or I still need to use UV Vis?
Usually, around 30 cycles are used for an endpoint PCR. This amount of cycles is enough to amplify your DNA fragment. However, people don't go out to 40 cycles for end point PCR so the fragment can also get used for cloning and sequencing. Beyond 30 cycles, there is a greater chance of non-specific amplification and PCR errors. Many PCR master mixes don't need any optimisation. But you can buy PCR optimisation kits if you're doing something specific. Perform the PCR with these mixes and run the samples on an agarose gel to see the best band. There are also DNA staining products that use LEDs to see the bands. But if your lab only has Ethidium Bromide, you'll need a UV light box or imaging system. You can also run the DNA on a capillary-based gel system to see the bands if you're lucky enough to have access to one. I hope this answers your question. 🤓 Please let me know if you have any further questions.
![Jack Harlow - Hello Miss Johnson [Official Music Video]](http://i.ytimg.com/vi/Q86_nlRoIGw/mqdefault.jpg)
I found that so many articles/videos discussed the topic of PCR and QPCR in such excess that, as a beginner, I struggled to define the difference in such a way that I could understand it. Your video/s and delivery of information are commendable and I cannot thank you enough for your channel. Superb.
Thanks for your comment. 🤓 You're very welcome. I'm so happy you found it easy to understand. It's great to hear. 👍❤
That was PERFECT. PERFECT. Everything. Down to the last-minute details.
I'm so glad it helped you. 👍 Thanks for your comment. 🤓
I love when algorithm finally shows a usefull and interesting video after a long time searching for a good explanation. Thanks a lot for your amazing work here.
This video, as well as the videos on Sanger and NGS, are brilliant. This the best molecular biology content I have ever seen on RUclips. Thank you so much, you are so great!!!
That's so great to hear! 🤓 Thanks for taking the time to comment.👍
*Welcome to ClevaLab* - if you like the video, please give it a 👍 and subscribe so we can reach more people. Also, if you have any questions, feel free to ask in the comments.
Get a *PDF Summary* of this video here: www.clevalab.com/post/pcrandqpcr.
finally i understand what is a quencher , thank you so so so much
Great, that's good to hear. I'm glad it helped you. Thanks for your comment. 🤓
This is AMAZING!!!!!!! Helped summarize (in detail) a 50 minute lecture in just 7 minutes!
Thanks for your comment. 🤓 That's great to hear, I'm glad it helped you.
this is my favorite video about PCR thank you. you made it easier to understand
Thanks for taking the time to comment. I'm so glad it made it easier to understand. 🤓
i love this!!! so easy to understand and the animation is super helpful thanksss
🥺💗
Great, I'm glad it helped you. 🤓 Thanks for your comment.
Wow. Yiu have earned a subscription
So clear and well explained
Thanks for subscribing. 🤓 I'm glad you enjoyed the video.
Somehow after a week of my professor trying to explain this to us and failing, this video helped me understand it in 7 minutes.
It's great to hear it helped you. 🤓 Thanks for your comment.
Love it! I have a question, I get that the elongation starts with the primers, but how does it end? Is there an end sequence or something like that?
this is such a good video. cant get a better explanation of PCR and qPCR than this video
Thanks so much for your comment. 🤓 I'm so glad you found it useful.
Thanks for designing a wonderful video. you made it easier to understand PCR for beginners.
Thanks for your comment. 🤓 So glad to hear the video made it easier to understand PCR. 👍
I recommend this video for novice in pcr
I seriously love your videos. Thank you
Thanks for your comment. 🤓 You're welcome, I'm glad they're helpful.
The most useful vid i’ve ever watched :)
Thanks for your comment. So glad you like it. 🤓
0:59 I remember One Direction ❤
This is an excellent PCR overview. I do have one question I was hoping you could answer. When selecting the primers for PCR, some textbooks/websites show the primers binding to the sequences flanking the DNA sequence of interest while others show the primers binding to the very beginning and end of the sequence of interest (not the flanking sequences). Can you explain the reason for this? I thought the primers always needed to bind to the "outside" of the sequence of interest?
Thanks for your question. 🤓 It can depend on the type of PCR you're doing. If you want to clone a specific gene or part of DNA, you will design the primers outside the region of interest. However, if you're doing gene expression, you'll choose a primer within the area of interest, usually across an intron boundary in the mRNA sequence.
Primer design might be a good topic for a future video!
@ClevaLab So it basically depends on what application you're using the PCR for. That's kind of what I was thinking, but I wanted to ask anyway. Thank you for your response, and I think it would be great to do a video on primer design in the future!
Amazing video. It helped me a lot!
Glad you liked it! 🤓
Well explained
I have one query.. Primer binds to strand and then it extends but how it's extension stops upto the end of particular gene. How it comes to know this is the end of gene rather continuing extension. Please reply
This was excellent. Thanks.
Thanks for your comment. 🤓 I'm glad you liked it.
Thanks for the video! many other videos on youtube but yours is easier to understand.
So glad you liked it, that's great to hear 👍🤓
Very helpful.I will like a video on Abbott RealTime PCR
Thanks for your comment. 🤓 OK, I'll keep that in mind. I have more PCR-themed videos planned for the future.
thanks so much for such a fantastic easy and simple explanation this makes my tomorrow paper preparation more easier🤩
You're very welcome. 🤓 I'm so glad it helped, thanks for taking the time to comment. 👍
I really enjoyed watching this 😃
Thanks for your comment. 🤓 So glad you liked it.
Well done
Fantastic
Thanks a lot. Glad you liked it. 🤓
I have a question for qPCR.
How many fluorescent atoms are in the probe repoter molcule? only one or several?
Thanks for your question. 🤓 For each DNA probe just one dye molecule is present. The most common dye is 6-FAM (Fluorescein), one 6-FAM molecule is added to each probe. To see the chemical structure see this link: en.wikipedia.org/wiki/6-Carboxyfluorescein
Thank you so much!
Thanks for your comment. 🤓 I'm glad you liked it.
Thank you!
@rezarezanaghibi You're welcome! Thanks for taking the time to comment. 🤓
great videos and channel
Thank you! 🤓
Please keep doing this
Thanks for commenting. 👍 There's another video in progress now. 🤓
Helpful! Thank you!
Thanks for your comment. So glad you found it helpful. 🤓👍
So, how do we make these very specific primers, which enable us to 'PCR' one specific gene, if we dont already know the DNA sequence? The sanger method of DNA sequencing, requires specific primers, complementary to the DNA younare trying to sequence. Well hold on a minute, thats a bit of a paradox isnt it. How do you know what the primer should look like if you dont know the sequence of DNA?
Thanks, good question. 🤓 The original method by Sanger to sequence PhiX174 (a bacteriophage), the first full-length sequence made, did not use primers. This was because primers were hard to generate then. Instead, they used restriction enzymes to cut DNA into fragments and used these fragments as the primers for their sequencing reaction. So, they didn't need to know the sequence as they depended on restriction sites to create the first full genome sequence.
Restriction enzymes are made by bacteria as a defence against bacteriophage (a virus for bacteria). They cut up the bacteriophage DNA to restrict its ability to reproduce. This fact was used by Sanger in combination with radiolabeled nucleotides.
Today, if a sequence is unknown, you can use random hexamers to amply it in a PCR or perform shot-gun or full genome sequencing without knowing any sequence. Short DNA fragments of known sequences are stuck on the ends. These known DNA fragments are then used to sequence the unknown DNA.
@@ClevaLab Thank you
amazing video
Thanks for your comment. 🤓 Glad you liked it.
thank you a lot; explanation with your animation is excellent and the flow ask for more......seven minutes viewing seems like 2 mins. .. thank your lots of work of animation behind for sharing knowledge🤩
I'm so glad you liked the animation and found it easy to watch and learn from. 🤓 Thanks for taking the time to comment. 👍
Awesome! Thank you, this is very helpful.
You're welcome! 🤓 I'm glad you found it helpful.
great video! Thanks! :)
Thanks for your comment. 🤓 So glad you enjoyed it.
THANKS
Thanks for your comment. 🤓 I'm glad you liked it.
Amazing
Thanks for your comment. 🤓
Great vedio ,it just reminds me of kinda animation of @amebasisters .
Thanks for your comment. 🤓 I choose to take that as a compliment.!
@@ClevaLab 🙂😂
Where's the 72°C for Taq polymerase?
Thanks for your comment. 🤓 That's a fair point, I should have included it, I can't remember why I didn't!
These videos always miss a very important fact that nucleotides are free-floating around with the rest of the slurry, and are snatched up by the DNA-polymerase as they pass by in order to be used for the copying.
Thanks for your comment. 🤓 That's a fair comment. I could have made that clearer in the video and had some nucleotides floating around.
Amazing! ❤
Thanks so much for your comment. 😁🤓
Wow just really fixed the matter for me, thanks!
Can I use the pictures for a presentation I need to show in class?
Thanks for your comment. I'm so glad it helped you. 🤓 Sure, you can find images in the blog post or even download a pdf. Find it here: www.clevalab.com/post/pcrandqpcr
@@ClevaLab thanks!
great vid love u
Thanks for your comment. 🤓 I'm glad you like it.
Why conventional PCR is qualitative and real-time PCR is quantitative? :o
Nice video, tho. Already subscribed. :)
Hi Abby, thanks for your question; it's a good one. Standard PCR can be semi-quantitative. We used to used this method in the lab before qPCR was available. Suppose you stop the PCR during the exponential growth phase of the samples. Then, you can compare the DNA level between samples. But, the dynamic range is tiny in comparison to qPCR. So these days, people use standard PCR for yes/no type testing.
I hope this helps and answers your question.
Please let me know if you have any further questions. 🤓
Thanks, so glad to have you as a subscriber. 🤓👍
@@ClevaLab Ohh, but in what phase in the amplification determines the "yes" or "no" answer? Is it in the plateau phase?
Also, if I were to optimize a PCR reaction, is it okay if I use Agarose Gel to determine the quality of DNA result through crowning? Or I still need to use UV Vis?
Usually, around 30 cycles are used for an endpoint PCR. This amount of cycles is enough to amplify your DNA fragment. However, people don't go out to 40 cycles for end point PCR so the fragment can also get used for cloning and sequencing. Beyond 30 cycles, there is a greater chance of non-specific amplification and PCR errors.
Many PCR master mixes don't need any optimisation. But you can buy PCR optimisation kits if you're doing something specific. Perform the PCR with these mixes and run the samples on an agarose gel to see the best band. There are also DNA staining products that use LEDs to see the bands. But if your lab only has Ethidium Bromide, you'll need a UV light box or imaging system. You can also run the DNA on a capillary-based gel system to see the bands if you're lucky enough to have access to one.
I hope this answers your question. 🤓 Please let me know if you have any further questions.
karry mullis has similar fate with Einstein :)
:)