cDNA Synthesis Protocol by Reverse Transcription
ฝัง
- เผยแพร่เมื่อ 14 ก.พ. 2021
- Hey Friends,
cDNA (complementary DNA) is synthesized from an RNA template. This is done by Reverse Transcription. In this example, the oligo d(T)-primers are used to prime the cDNA synthesis. An alternative would be to use random hexamers as primers. cDNA is synthesized before qPCR to check gene expression levels.
qPCR:
• qPCR (real-time PCR) p...
References:
Recommended: www.nature.com/articles/nmeth...
More:
geneticeducation.co.in/revers...
www.ncbi.nlm.nih.gov/pmc/arti...
Cheers
Henrik - วิทยาศาสตร์และเทคโนโลยี
I was so confused with these terms and you explained so clearly and so well. Thank you so much
Thank you soooo much for this.
This thing was more like rocket science to me before watching this video. Cleared all the concepts in just one go.
Thanks again. 😊
Have great day ahead
Merci pour la vidéo, elle est très claire et parfaite pour bien comprendre le mécanisme de la retrotransciption !
Thank you !
Thank u so much. before I watch this video I read some what notes but they were boring. ur video were so interesting with smooth sound and full information.
Thank you we need more explanation in different Lab techniques. It is practical and we know need to know the sciences. Most of the time I find myself confused in class but thanks to you now I understand it better. Keep it up we need you. You deserve all the success.
Sehr verständlich, vielen Dank!
Thank You So much Sir.. I watched lot of Videos But No one explained about RNaseH Function and I was really confused. You Really Explained very nicely. Thanks for making this video..
Excellent explanation & graphics.
Keep up the good work!
Thank you so much, Sir! the explanation is to be so clear and easy to understand for us.
Sir
I have lack of word to thanks you.
Really you I m very excited after watched this .
Stay bless forever God bless you More and more forever ❣️💝
This is great. Perfect rythm. Thank you.
Very helpful! I finally understand what do we actually use reverse transcription for, thank you!
Excellent video. You made this concept easy to understand, thank you.
Embrace tranquility. Thanks, Zen
Thank you so much. The explanation is very intelligible.
Fantastic! Amazing! Incredible! Astounding! Mighty! Wonder! Super!
Wow
Very good explanation man. Thank you so much!
Excellent explanation!
Thanks you so much finally got what i was looking for
Very well explained concepts are clear about cdna
This helped me with my genomics class 🔥🤙🏿
i was confused for intron thingy and u made it easy
Great explanation!
Thank you so much Henrik's Lab. You are doing a very noble job by providing us such useful concept clearing video. Through your videos we are able to understand the topic better than hours of classroom learning. ❤❤
Very pleased to read that! Thanks ❤
At 2:02 , that mrna and dna combination could contain all 5 types of nucleotide, A,T,G,C,U?
Yes @@rajmangalpandey3910 you are right. The mRNA-cDNA hybrid so produced contains all 5 bases.
Please also note that only the template mRNA strand contains Uracil.
The two enzymes used here are:
(1) RNA dependent DNA polymerase (reverse transcriptase) and
(2) DNA dependent DNA polymerase (DNA polymerase 1)
These enzymes used here cannot add uracil during polymerization, therefore uracil will be absent in the newly synthesized DNA strands.
@@Abhinav-kf4wd why Rnase didn't ate whole RNA? But made small fragments
@@rajmangalpandey3910 I do not know the reason.
But If RNase H hydrolyzed the entire mRNA strand then there would be no primer left for DNA polymerase 1 to act.
Amazing! Thanks!
Keep up the good work
amazing explanation. cheers mate
Muito bom o vídeo, parabéns 👏🏽👏🏽👏🏽💚
Muy buen video! Gracias!
thank you very very much!
Thank you 😊💕💕
Thank you so much sir❤
it is very nice and please adding brief explanation about recombinant DNA
amazing , keep going
Thank you very much..
Nice explanation ❤️
Thank you so much!
Thank you ❤ God bless you ❤️
Best video ever❤️❤️
How do you make cohesive ends on the cDNA so you can be able to ligate it with the chosen vector?(using restriction enzymes for the cDNA given would cut out some of the coding sequence right? so how do you add cohesive ends? thanks
🙏thank you.
Thanks!!!!
Thank you
Hey, one question after the synthesis of ds-cDNA. How it will multiply in numbers.................Without having the primer for coding another strand....?
Thanks :)
Hi, i hope someone will help you the way u helped me today, I wish u the best from France
My doctor should watch this srsly
Danke, brudi! Arbeiten grade an einer präsi über Gen Chips und das ist verdammt nützlich!!! Love you
Hello, i am facing problem in cdna synthesis. would you please help me to know your protocol?
At 3:20 the last exon shouldn't have blue "legs", because blue was polyA before. Poly A is not in DNA.
Mam 5'AGUCAGGUC3' this is mrna what is the C dna to this sequence
Today, my Professor explained this topic to us, somehow she explained, that after the synthesis of the first cDNA-Strand the Double-Strand would be denaturated into Two seperate ones, later there are inhibitors to Stopp the accurance of Rnase. Later Taq-Polymerase is Synthesizing the other Strand from the cDNA-Strand that was reverse trnascripted before, not like in your video by Nicking from the Rnase and Polymerase 1 and later with Ligase. I asked her about this and she thought that you explained it as an example how it would happen in the body not like in Biotechnology. Im got super confused that she explained it different at some point. Weirdly enough she suggested us your video to learn the reverse transkription, thats why i confronted her today, in order to get clear what is correct for her exam XD
Maybe the is some potential to make a different video out of it?
Danke für das gute Video. Hab es dennoch gut verstehen können!
Hey,
thanks for this message! I created this video based on the following nature paper: www.nature.com/articles/nmeth0205-151
Here, it also includes the nicks. But as alwayas: there is not only one protocol or kit. There are a few ways to generate cDNA. Hope it helps a little!
Is there no hairpin loop?
in 2:57 may I ask how whole mrna are replaced by the DNA? thank you
I think, the same exonuclease activity will remove the rest of the RNA parts
Thank you. How can we contact you if we get problem while doing qpcr wirk?
Either here or in the comment section of the qPCR video
Thank you for your quick response. Do you have some vedios for analyzing the data of qPCR?
@@user-mm8cq9xg3m not for data analysis yet.. but I indeed consider to do one (end of April)
Why cant we use mRNA directly in microarray or qPCR
it wont express completely to give information. we must use reverse transcriptase, bcos hybridization will only occur when we will use cDNA due to presence of oligo sequences on microarray.
Wow! what can I get you from the vending machine
Is there a way we could translate all the proteins in a human genome and create a lab created human since the entire genome has been sequenced?
Hope I got your point (create a completely synthetic human with proteins that we put in cells)? Very interesting idea. I know that something like this was done already with bacteria... but for mammals this would be way more difficult. Image, there are more things than protein.. regulatory RNAs, the DNA itself is required.. there is methylation (some genes are inactivated and some others activated). So there are more components than protein, but extremely interesting to follow up on this in a smaller way!
😃😃
I have a question. What´s the meaning of the last product in this process? what is "ds"?
Thanks for asking!
dsDNA = double stranded DNA
@@henrikslab Thanks for supported me! Your video is all that i needed to understand this process. SUPER LIKE :)
Can you Translation.. to Arabic
Bist du deutscher ? So hörst du dich an XD
deutsch wär echt cooler gewesen
Wait a second people...forgive my lack of understanding but i dont understand why start to syntettize anything about and HUMAN DNA??? Why want to know what GOD did once and why and how and how to REVERSE IT/////?????!! Please someone explain..Many thanks
Only virus can do that
We can do literally anything by manipulating our genes. Ofcourse, to do so, one needs to know how to speak the language of life; the language of proteins. Our genes controls our everything. You can be eternally healthy, have a perfect immune system, become a superpowered human, have 200 IQ, be able to photosynthesize, and literally become biologically equivalent of immortal.
We reverse it to recreate it, and improve it.
🤣🤣🤣🤣
Bruder sprich doch deutsch
Man trying to play God not our place to mess with God's code that makes us human.
I've been reading about the cDna lately after the vakka vakka
I can't quite decipher it yet
th-cam.com/video/RAYZtXMEowo/w-d-xo.html
Just look for the explanation
I come across this cDNA when someone takes the Weuka Woeka
As in the days of Noah !!! Praise the Lord !!!
Did you know that RNA isn’t just a human thing?
We use this process for a very useful purpose at our citrus pathology lab. It helps us to see if our treatments against a particular pathogen are useful or not.
Shut up you talk to the sky like a crazy person