Excellent summary. I've been doing this for years, and I'm not sure if I ever thought about why the probe isn't extended like the primer is, but the 'not free -OH group' explained it. Thanks
This is a great video, simple, clear and very well designed. The explanations perfectly fit the images. I personally like the enthusiasm of the narrator. Thanks for the help!
thanks for the video, was so easy to understand. Do you have one video to explain gene expression assay by taqman?, that will be very helpfull. THANKS from Ecuador (south America)
Thank you! In a perfect world, every scientist working in the field today would still recall that optimism and enthusiasm they had for science when they first discovered it.
Thanks for this very clear and compact explanation. Just one addition: The "F" in "FRET" stands for "Foerster" (the guy who initially described the phenomenon), not for "fluorescence".
Thank you so much! This is the very best explanation for taq man! Short and clear! like all of your videos. You helped me so much to set up a good "methods" part in my master thesis ;) greetings from germany!
Thank you so much! I'm studying for an exam and I didn't fully understand the principles but the way the information was presented in this video is amazing! I have a better understanding now :)
Very good video! I thank you as a german student, because I need this for a practical course where we use it and have to write a protocol. Wish there would be more videos how something works. Even your english is clear and easy to understand. :)
Do you have a video explaining how the allelic discrimination work? By that I mean I'm trying to understand how you take the allelic discrimination plot chart after the PCR instruments spits out the result to understand what your data means. I hope you understand what i'm asking?
In the notes about the Life Technologies TaqMan assays, it is said that primer efficiency can range from 90-110%. How can a primer pair be more than 100% efficient? What does this mean?
how to setup the applied biosystem ONE-step instrument for performing SNP genotyping (step by step)? please help me to do it from plate setup to start run
how to setup the applied biosystem ONE-step instrument for performing absence/presence (step by step)? please help me to do it from first step of plate setup to last step start run
I love how this guy explained everything. Thank you sir. Made my assignment a whole lot easier. Can you do a next video on LAMP assay? --Dr.Rakesh Chand from Infectious Disease Centre, Tokyo University of Agriculture and Technology.
In the case of SNP mapping, a stable duplex forms between the probe and the segment of interest only if the probe is perfectly complimentary to the DNA sequence. If there is a SNP, incomplete binding occurs and the probe is not cleaved. My question is, how does one determine where exactly on the segment of interest is the SNP?
Your scenario sounds more like SNP mapping using sequencing. With a TaqMan SNP assay, the genome sequence (and SNP) is known. Two probes are designed to the SNP site, one probe to detect each allele, labeled with either VIC or FAM.
I am a mechanical engineer I am not a biologist. I'm trying to get my head around how PCR can actually benefit mankind. I understand the part about growing strands of DNA. Is PCR purely a biological notion? Or can it do things like build a bridge or produce milk at a faster rate?
I have a simple question, as a newbie to this technique; In the same reaction environment, how close are these probe molecules and all the cycle products to each other that they do not quench all the freed fluorescent molecules in the vicinity? Afterall, everything is in a single and crowded reaction tube.
Dear friend, To run this assays, can I use normal PCR reagents, like normal DNA TaqPol? Of course it is necessary the primers forward and reverse and the two specifs probs. But how about the others reagents?
If I amplify a genomic region in a heterozygous species I will potentially have two PCR products in one reaction, but I may not be sure which ones. Is there a way I can pool several designed TaqMan probes (each designed for a certain haplotype, and maybe up to 10-20) to discover what SNPs (and therefore what haplotypes) exist in a certain sample? I suppose each probe would need to fluoresce a different color or intensity. Essentially I am asking if I can use more than two TaqMan probes per reaction to genotype an individual.
hi.I am doing genotyping using GTXpress master mix and was wondering if I could use a total reaction volume of 5 microlitres (instead of 10) using 2.5 microlitres of master mix ( i am on a low budget!). I ve tried it once and it seems to work fine! thanks!
We have not validated that workflow for a 5uL reaction, however since the genotyping assays are qualitative (not quantitative), you can probably get away with it.
Hi, why do we normalize genes? I do understand that it the housekeeping gene effectively monitors and detects the relative expression of cDNA from sample to sample, allowing us not necessarily use the same amount of concentration when doing qPCR of multiple samples. Can someone go a little more in depth on this? Thank you!
Hi Nagarjun. Thank you for your question. Our imaging system is able to read the fluorescence that is given off of the well and collect the necessary data. It records as fluorescence units. Regarding your units in India question, we recommend that you contact us at thermofisher.com/askaquestion so that your inventory question can be routed to the team in India. Thank you!
Great question! Using two probes in the same well would be a duplex reaction. When multiplexing like this, you would always want to validate first to make sure there are no probe or primer interactions. This means you would compare the results when the assays are run in single wells (singleplex), versus combined into the same wells (multiplex). If there are no interactions between the probes (or primers) then the Ct values will not change in the two experiments, and it is okay to proceed with the multiplex experiments. In a singleplex reaction, the primer and probe sequences go thru bioinformatic screening to make sure that they are specific and checked for inter and intra sequence interactions. Check out our Application Note on our Design Process for more details: tools.lifetechnologies.com/content/sfs/brochures/cms_040599.pdf
+Amr Halawani Thanks for the feedback, Amr! To answer your question: Yes. It uses the same technology as TaqMan assays. The probes are specific for Ion Sequencing adaptors.
When do you qPCR, the master mix usually contains a mixture of uracils and thymines, meaning the qPCR product generated will contain a mixture of these bases. UDG/UNG degrades uracil-containing amplicons, and therefore serves to degrade amplicons generated during PREVIOUS runs, so that they don't contaminate your CURRENT run. The 50 degree incubation step before thermal cycling is where UDG does its work, then it gets in activated at the 95 degree step so it cannot degrade new uracil-containing amplicons that are about to be made. Hope this helps.
Love the explanation so much!! great video!
Thanks, we are glad you enjoyed the video.
This man is so happy to be alive :))))))))))
This is the first time I see a video of Taqman, but I am already considering a fanclub.
Oh, boy, this man LOVES science! Hahahaha
Your comment cracked me up! Epic!
I understood in 3:59 minutes what I've been trying to understand for hours on the internet. Thank you!
That's amazing to hear, thank you!
So charismatic!!! Brilliant explanation!!
This is an *Incredible* video that shows with ease and very clear simple animations how TaqMAN works.
Great Job!
Easily understandable explanation and good-looking animation. A thank you from a German student.
Trafficlight
Please tell Me in words how this technology is better the Dan real time pcr.
Excellent summary. I've been doing this for years, and I'm not sure if I ever thought about why the probe isn't extended like the primer is, but the 'not free -OH group' explained it. Thanks
Aw, this guy is cutest!
But in seriousness - very concise and informative! Thank you for the explanation :)
This dude has such an infectious enthusiasm for this stuff, no pun intended
thank you. short and crisp explanation!
I am so impressed by the clarity of his explanation. Thank you!
Thanks Blair! Let us know if we can answer any questions!
This is a great video, simple, clear and very well designed!!! also love the enthusiasm of the narrator!!
I don't know why but listening to this man is making me happy
This is a great video, simple, clear and very well designed. The explanations perfectly fit the images. I personally like the enthusiasm of the narrator. Thanks for the help!
I love how you explaining everything with enthusiasm!!
Your energy was fluorescenting throughout the whole video - love that.
Science can be Taqsing on the mind and body - a true, unadulterated enthusiasm for the work goes a long way.
You are The greatest teacher i have ever seen...!!!
Such an infectious presenter. Love it!
This video made my life way easier! thanks to you TAQMAN!!!!
Great job! Love the enthusiasm and ease of understanding this process!
Not everything in science is as easy as we'd like it to be! We're being the edutainment we want to see in the world.
Thank you guys! Greetings from Greece!
thanks for the video, was so easy to understand. Do you have one video to explain gene expression assay by taqman?, that will be very helpfull.
THANKS from Ecuador (south America)
Very enthusiastic, made the video enjoyable to watch.
The best ever lecture on Taqman probe.....Thank you❤❤
Great video. Your enthusiasm makes it so interesting!
You're never too old for edutainment! That's our (unofficial) policy.
@@thermofisher 😍
I FINALLY understand how TaqMAN works!!!!! Thank you. Loved every bit of the enthusiasm!
Thank you! In a perfect world, every scientist working in the field today would still recall that optimism and enthusiasm they had for science when they first discovered it.
Thanks for this very clear and compact explanation. Just one addition: The "F" in "FRET" stands for "Foerster" (the guy who initially described the phenomenon), not for "fluorescence".
Thank you so much! This is the very best explanation for taq man! Short and clear!
like all of your videos.
You helped me so much to set up a good "methods" part in my master thesis ;)
greetings from germany!
+Lara Kuerzer Thanks, Lara! We appreciate the feedback and let us know if we can answer any questions for you. Good luck on your thesis!
Hi lara
Perfect for my MEDSCI 203 paper!! Thank you from New Zealand :)
Well presented! I love this video so much!
Great explanation, and the enthusiasm about the topic is awesome!
YOU'RE AWESOME TAQMAN!
This was very helpful and your enthusiasm was wonderful! Thank you
neeey friends ! not taq polymerase !! haha
I just looove the way you explain and make the science funn ! :D
Brilliant explanation. I like the enthusiasm he exudes. Thanks you.
Thank you so much! I'm studying for an exam and I didn't fully understand the principles but the way the information was presented in this video is amazing! I have a better understanding now :)
Great explanation!!! Greetings from The Gambia.
Very good video! I thank you as a german student, because I need this for a practical course where we use it and have to write a protocol. Wish there would be more videos how something works. Even your english is clear and easy to understand. :)
AMAZING! THANK YOU SO MUCH!
Love the explanation fantastic
Full of energy! Do more videos like this for biotech. You earned my like
We will do our best!
Do you have a video explaining how the allelic discrimination work? By that I mean I'm trying to understand how you take the allelic discrimination plot chart after the PCR instruments spits out the result to understand what your data means. I hope you understand what i'm asking?
Such a good video! Tnx for the explanation!
Our pleasure!
So, why do we need both forward and reverse primers, if the probe only attach to one strand?
You have a way of making science interesting, Thanks for the video
Very, very elucidative! Thank you!
This video is extremely helpful, thank you!
In the notes about the Life Technologies TaqMan assays, it is said that primer efficiency can range from 90-110%. How can a primer pair be more than 100% efficient? What does this mean?
how to setup the applied biosystem ONE-step instrument for performing SNP genotyping (step by step)?
please help me to do it from plate setup to start run
Great video!!
how to setup the applied biosystem ONE-step instrument for performing absence/presence (step by step)?
please help me to do it from first step of plate setup to last step start run
Hahaha Made my revision much simpler! Thanks :D
Thank you, this is very helpful
You're very welcome, Bryan. Let us know if we can help with anything.
I love how this guy explained everything. Thank you sir. Made my assignment a whole lot easier.
Can you do a next video on LAMP assay?
--Dr.Rakesh Chand from Infectious Disease Centre, Tokyo University of Agriculture and Technology.
Perfect video, the explanation is detailed and easy to understand! Thank you!
Our pleasure! We're glad you found it so useful!
In the case of SNP mapping, a stable duplex forms between the probe and the segment of interest only if the probe is perfectly complimentary to the DNA sequence. If there is a SNP, incomplete binding occurs and the probe is not cleaved. My question is, how does one determine where exactly on the segment of interest is the SNP?
Your scenario sounds more like SNP mapping using sequencing. With a TaqMan SNP assay, the genome sequence (and SNP) is known. Two probes are designed to the SNP site, one probe to detect each allele, labeled with either VIC or FAM.
I am a mechanical engineer I am not a biologist. I'm trying to get my head around how PCR can actually benefit mankind. I understand the part about growing strands of DNA. Is PCR purely a biological notion? Or can it do things like build a bridge or produce milk at a faster rate?
you helped me so much. THANKS from germany :)
very enthusiastic guy
thank you!! great presentation!!
This was really helpful. thank you
Your videos are awesome.
fantastic
I have a simple question, as a newbie to this technique; In the same reaction environment, how close are these probe molecules and all the cycle products to each other that they do not quench all the freed fluorescent molecules in the vicinity? Afterall, everything is in a single and crowded reaction tube.
Dear friend,
To run this assays, can I use normal PCR reagents, like normal DNA TaqPol? Of course it is necessary the primers forward and reverse and the two specifs probs. But how about the others reagents?
If I amplify a genomic region in a heterozygous species I will potentially have two PCR products in one reaction, but I may not be sure which ones. Is there a way I can pool several designed TaqMan probes (each designed for a certain haplotype, and maybe up to 10-20) to discover what SNPs (and therefore what haplotypes) exist in a certain sample? I suppose each probe would need to fluoresce a different color or intensity. Essentially I am asking if I can use more than two TaqMan probes per reaction to genotype an individual.
Thanks! This is so helpful!
Wow ! a pleasant presentation
Give that man a promotion!
Understood it really well
great ! best explanation found . Thank's a million
thank you loads for the things you are explaining...that's really helpfyul!
Happy to help! We enjoy spreading scientific knowledge and insight wherever we can!
wow, wonderful explanation thanks
Great! I would like to know which is the best real time PCR machine?
hi.I am doing genotyping using GTXpress master mix and was wondering if I could use a total reaction volume of 5 microlitres (instead of 10) using 2.5 microlitres of master mix ( i am on a low budget!). I ve tried it once and it seems to work fine! thanks!
We have not validated that workflow for a 5uL reaction, however since the genotyping assays are qualitative (not quantitative), you can probably get away with it.
Thank you so much for your invaluable explanation.
We're glad to hear it was useful! Thanks for watching.
Hi, why do we normalize genes? I do understand that it the housekeeping gene effectively monitors and detects the relative expression of cDNA from sample to sample, allowing us not necessarily use the same amount of concentration when doing qPCR of multiple samples. Can someone go a little more in depth on this? Thank you!
How do you log the Real time data that you receive from the fluorescent light? And how many units do you have in stock in India?
Hi Nagarjun. Thank you for your question.
Our imaging system is able to read the fluorescence that is given off of the well and collect the necessary data. It records as fluorescence units.
Regarding your units in India question, we recommend that you contact us at thermofisher.com/askaquestion so that your inventory question can be routed to the team in India.
Thank you!
thank you very much
this video helps a lot
What prevents two probes from binding to one another and distorting the quantitative values?
Also, this video was extremely helpful, great work at simplifying something that can be very confusing when read only in text!
Great question! Using two probes in the same well would be a duplex reaction. When multiplexing like this, you would always want to validate first to make sure there are no probe or primer interactions. This means you would compare the results when the assays are run in single wells (singleplex), versus combined into the same wells (multiplex). If there are no interactions between the probes (or primers) then the Ct values will not change in the two experiments, and it is okay to proceed with the multiplex experiments.
In a singleplex reaction, the primer and probe sequences go thru bioinformatic screening to make sure that they are specific and checked for inter and intra sequence interactions. Check out our Application Note on our Design Process for more details: tools.lifetechnologies.com/content/sfs/brochures/cms_040599.pdf
Scott Johnson
And thank you for the feedback. We'll be making more videos soon so stay tuned!
Awesome explanation but for SNP detecton, would you use a singleplex assay or multiplex assay?
This is an amazing explanarion! I got a nice laugh out of the Pac Man tie in. Very well done!
this is great thanks a lot!!!
+daman kaur You're very welcome and thanks for watching!
Joining my fellow grateful students of past, present, and future in this comment section haha, good video, thanks for explaining it so well!
Our pleasure! Thank you for taking the time to watch this.
very very useful!!
Thanks for watching.
Thank for your explanation, it very help me to understanding q PCR
Our pleasure! Thanks for giving us a look.
@@thermofisher yure welcome
man youre a legend!!
thank you, that was really helpful
Thank you for watching!
this is a very helpful video.
We're glad you like it! Thanks for giving it a watch.
Very worthwhile explanation! Is this the same technique for the Ion Quantitation kit that is used to quantify NGS libraries?
+Amr Halawani Thanks for the feedback, Amr! To answer your question: Yes. It uses the same technology as TaqMan assays. The probes are specific for Ion Sequencing adaptors.
Oh man! I would like to be like you...
you saved me, thank you!
what is the real name of this scientist/actor?
+Sérgio Rocha His name is Doug Rains.
Well explained Sir
Thank you! Hope you found it useful and engaging.
great!!
God bless you 🙏
what is the role of UDG in taqman quantification kit?
When do you qPCR, the master mix usually contains a mixture of uracils and thymines, meaning the qPCR product generated will contain a mixture of these bases. UDG/UNG degrades uracil-containing amplicons, and therefore serves to degrade amplicons generated during PREVIOUS runs, so that they don't contaminate your CURRENT run. The 50 degree incubation step before thermal cycling is where UDG does its work, then it gets in activated at the 95 degree step so it cannot degrade new uracil-containing amplicons that are about to be made. Hope this helps.
korean ver plz
I finally get it now
Thanks for watching.
This is amazing thxxxx