I have a final on the Dec 19th and I'm going over all the topics that were a little confusing, boy have you cleared alot of them up. Thank you so much, I'm not sure I could've figured all of this out in 16 min. I wish our education system had professors like you.
@@AKLECTURES I only waschen your lectures! Not my Professor! I not only get frustraded in his lectures! But also feel so very overwelmed! When I Watch your lectures I feel happy and confident, and love science ! It's like watching a favourite show that also gives us good grades! 😁
Iam a student of Pharmacy and Biochemistry in the University of Buenos Aires. Thank you very much for your classes. Sanger Sequencing of DNA part I and II was very usefull , among other subjects as well Melting DNA and .... Greetings from Buenos Aires Argentina
Thanks for the amazing lecture. You explained as simple as possible. It is extremely easy to follow and clarified some areas I didn't grasp right away. Im sharing this link with my genetics class!
How do you determine the sequence of the complementary nucleotides for the designed primer before sequencing the template strand if the sequence of bases on the template is still unknown at this stage?
Piotr Klimczak If you don't know any base sequence of the template, you can use the base sequence of the restriction site of the Plasmid (from your library) as the starting sequence, which is then followed by the DNA fragment you want to sequence.
sometimes they add an adaptor kinda molecule onto the 5' end of your DNA. the adaptor was designed by you so you'd know the primer to use. for example it can we a poly A molecule. so your primer would be poly T. and the actual DNA can get sequenced from the very first nucleotide.
Hey dude. Great lectures. one question. How do you know what your primer sequence is? Do you know what it is because you know what your vector sequence is at the beginning and end of you MCS?
thanks very much Sir. I have one questions please. while using the dNTPs ( dATP, dGTP, dCTP, TTP) why used TTP instead of dTTP like others?? I am very thankful for your efforts...
since it is already implied that Thymine is only used in DNA (characterized by the "deoxy or D" prefix) and not used in RNA, so by convention we drop the deoxy prefix as we no longer need to specify the difference. In the other ones we keep the deoxy prefix to make the distinction between the NTPs and DNTPs
Technically we use terminal transferase for addition of poly N tail (N being any nucleotide- A/G/C/T) to the 3' end then we can easily add a radiolabelled complementary poly tail.
Great video! I have one question though: how do they determine the primer sequence, like how do they know the complementary sequence of the primer that they should make?
I have a question in regards to Sanger Sequencing. Sequencing of the DNA fragments will stop at all possible positions where the particular dideoxynucleotide is incorporated before starting a new chain and proceeding to the next ddNTP. How is this ensured exactly? How do we not end up with whole lot of random sequences or all short fragments or all really long fragments of DNA for each ddA, ddT, ddC and ddG? I hope my question is clear, Thank you.
I have few queries please help me out: 1. If this DNA is unknown then how is primer designed? 2. In each tube there must be a DNA sequence which is formed from only dNTP's and no ddNTP, so in gel electrophoresis each lane will have a full size DNA, eg- if DNA length is 10 bases then in each tube there must be a replicated DNA of length 10 formed by all dNTP's then in this case how do we know the sequence of the last nucleotide after gel electrophoresis?
Hi , its great video dude. May I know why this synthesis have to choose ddATP ( it stops when choosing). How does it happen? If I m not wrong, this happens randomly that helps us to get particular sizes that make the sequence in order when running electrophoresis. Pls return if Im not correct. Thanks
Plz answer if you know : Would it be possible that because of low concentration (0.01), the enzyme does not use ddNtp in all probable part to stop replication and as a result we miss one fragment possibility?
how many single-stranded DNA molecules are we putting into those beakers? and.. what if we put too much DNA strand molecules and the ddNTP and dNTP have been used up ( causing repeated base-pairings of the same mass) before the template strand can be sequenced completely?
If you're using a 100:1 ratio of ddNTPs, how will you sequence the part of the DNA which are longer (travalled the least), the region before the ddNTP?
What happens if we get a full length molecule in the gel electrophoresis...let say if no ddATP was pick to make the complementary DNA molecule in beaker no.1 ...then we obtain a full length molecule plus the 3 shorter molecules. Is this full length molecule runs into the gel too? If so what happens for the reading of the gel electrophoresis?
My question is: when the two strand of DNA molecule are separated, how do we do to make sure that in the beaker, we will have only one of the two strand in this beaker. How can we be sure that there is no contamination with the other strand that we do not want?
Sir, I have a question that came in my exam and I didn’t know how to solve it he asked us if we placed a double strand while using sangers dna sequencing what will happen I couldn’t figure it out can u answer me please
Hi, I don't understand how you got 3 fragments with ddATP, when the DNA Polymerase adds ddATP the synthesis stop isnt it? so how the next fragments are obtained? I would appreciate your help!!! Thank you
I am from India sir and your lectures are truly helpful for me and i suggest others also to watch your lectures as u make the concept very clear and understandable with out any complexity. Each and every topic was very well explained and when ever i travel and don't take books along myself i prefer your lectures. I am very thankful of u sir, by seeing your lectures at my room it seems that i am attending lecture. Not before exams but also during my free time i watch your lectures to clear my concept and there is revision of basics also. Thank u so mch. Regards: Shantanu tomar
![[UA] NAVI vs MOUZ | BO5 | IEM Rio 2024](http://i.ytimg.com/vi/CMBYk0cbGoA/mqdefault.jpg)
spent 1 hour trying to understand the whole process from my lecture slide and this guy easily and properly explains it under 20 mins -
dude u just saved my life . i had to make a project about it . and i didnt undetstand a thing . thanks bro . thanks a lot !! now i get it.
You are just fabulous. I must say this is the best lecture on Sanger Sequencing on youtube..
I have a final on the Dec 19th and I'm going over all the topics that were a little confusing, boy have you cleared alot of them up. Thank you so much, I'm not sure I could've figured all of this out in 16 min. I wish our education system had professors like you.
One of the best discussions I had regarding DNA sequencing.... Nothing could be easier than this....
LET ME JUST SAY THIS!!! 😭I WAS SO CONFUSED ABOUT THIS PROCESS AND YOU BROKE IT DOWN SO perfectly!!! THANK YOUUUUUU SO MUCHHHHH
The best video ever has explained Sanger Sequencing 👌 … THANK YOU !
First year 12 bio test and this just answered my last query, very well presented!
My professor plays your videos in the lectures .. she does her job good lmao!! anyway THANK YOU !!
Ava S thats awesome! many thanks to your professor :-)
that is pathetic. The very least she could do is memorize his lecture and repeat it.
@@AKLECTURES I only waschen your lectures! Not my Professor! I not only get frustraded in his lectures! But also feel so very overwelmed! When I Watch your lectures I feel happy and confident, and love science ! It's like watching a favourite show that also gives us good grades! 😁
Iam a student of Pharmacy and Biochemistry in the University of Buenos Aires. Thank you very much for your classes.
Sanger Sequencing of DNA part I and II was very usefull , among other subjects as well Melting DNA and .... Greetings from Buenos Aires Argentina
LIFE SAVER!!!!! Made that so easy to understand!
seriously YOU ROCK !!!! thank you sooo much you've saved my life !!
Thanks for the amazing lecture. You explained as simple as possible. It is extremely easy to follow and clarified some areas I didn't grasp right away. Im sharing this link with my genetics class!
My lab professor left me lost as to how to read and understand this process smh. A 2.5 hour class beat out by a 20 minute UA-cam video! Gratitude sir
If I ever run into you I am buying you a beer. Thank you for making videos that are so clear, understandable and pure awesomeness.
You did such a great job at explaining this topic!! Thank you!!!!
you literally saved my life. easy and incredibly helpful explanation. my professors should take notes.
Best teacher I have ever seen in my life
You are a genius man. Thank you for the detailed presentation
How do you determine the sequence of the complementary nucleotides for the designed primer before sequencing the template strand if the sequence of bases on the template is still unknown at this stage?
Piotr Klimczak If you don't know any base sequence of the template, you can use the base sequence of the restriction site of the Plasmid (from your library) as the starting sequence, which is then followed by the DNA fragment you want to sequence.
sometimes they add an adaptor kinda molecule onto the 5' end of your DNA. the adaptor was designed by you so you'd know the primer to use. for example it can we a poly A molecule. so your primer would be poly T. and the actual DNA can get sequenced from the very first nucleotide.
thank you for your great lectures ! very useful and well explained!
You have extraordinary communication skill.
Excellent explanation 👌 I understand what you say one 💯 percent. Thanks alot
Really got crystal clear concept.... Thanks
thank you so much. this was well explained. You are great at this
this actually blew my mind, crazy stuff right there.
He is amazon. We need more videos like this from him.
You are the best. Ever.
Thank you so much. You made this so much easier to grasp and understand 😊
Hey dude. Great lectures. one question. How do you know what your primer sequence is? Do you know what it is because you know what your vector sequence is at the beginning and end of you MCS?
Best teacher ever!
thanks bro for very good lecture keep it up and always make these kind of videos ,
u will always be in our prayers .
you're videos are amazingly useful! keep going!!
That was such an awesome explanation thank you so much!
Great explanation , really it's very useful , many thanks
thanks very much Sir.
I have one questions please. while using the dNTPs ( dATP, dGTP, dCTP, TTP)
why used TTP instead of dTTP like others??
I am very thankful for your efforts...
since it is already implied that Thymine is only used in DNA (characterized by the "deoxy or D" prefix) and not used in RNA, so by convention we drop the deoxy prefix as we no longer need to specify the difference. In the other ones we keep the deoxy prefix to make the distinction between the NTPs and DNTPs
Useful video to help me understand about it, thanks a lot!
I really can't thank you enough 💜💜 You really helped me end my struggling with it .
Thank u I learn my biology from only your leactures
Thank you thank you thank you!!!!!
How does one figure out the primers needed for an unknown DNA sequence?
+Rafael Sebastián did u come to know, if u do, please state here .
+Gio Pietra how did they come to know that unknown sequence from scratch?
Technically we use terminal transferase for addition of poly N tail (N being any nucleotide- A/G/C/T) to the 3' end then we can easily add a radiolabelled complementary poly tail.
human genome project
i needed this kind of explation. life saver
You could simply have explained it in part1...why need to make part2 and btw that was very helpful...i appreciate it...tysm
Great video! I have one question though: how do they determine the primer sequence, like how do they know the complementary sequence of the primer that they should make?
Thank u alot, that was very helpful.
U are a great tutorial .
You are truly a legend 🤩
I'm very lucky to be your follower on UA-cam 😃
He seems so excited in this part.
Hi! I have a question about the Sanger sequencing, how do you isolate just one strand of the DNA to sequence?
Thx for the wonderful explanation!!
At last I understand it!!!! Thank you! very useful and clear : )!!!
great video !!! finally I understood the principle of sanger sequencing .-) thanks a lot !!!
I have a question in regards to Sanger Sequencing. Sequencing of the DNA fragments will stop at all possible positions where the particular dideoxynucleotide is incorporated before starting a new chain and proceeding to the next ddNTP. How is this ensured exactly? How do we not end up with whole lot of random sequences or all short fragments or all really long fragments of DNA for each ddA, ddT, ddC and ddG?
I hope my question is clear,
Thank you.
I have few queries please help me out:
1. If this DNA is unknown then how is primer designed?
2. In each tube there must be a DNA sequence which is formed from only dNTP's and no ddNTP, so in gel electrophoresis each lane will have a full size DNA, eg- if DNA length is 10 bases then in each tube there must be a replicated DNA of length 10 formed by all dNTP's then in this case how do we know the sequence of the last nucleotide after gel electrophoresis?
Great video. smooth and very clear!
Yvan Delgado de la flor Thanks! :)
Thank you so much 🥰
It's short et clear
Hi , its great video dude. May I know why this synthesis have to choose ddATP ( it stops when choosing). How does it happen? If I m not wrong, this happens randomly that helps us to get particular sizes that make the sequence in order when running electrophoresis. Pls return if Im not correct. Thanks
Can’t believe I pay much for tuition. This is outrageously succinct. Amirite?
You are amazing! Kudos!
Brilliant! Thank you!
Thank you sir... just what I was looking for!
Thank you for your lec.
Love your lectures!
+Evgeniya Fedorova Thanks Evgeniya!
legend, thanks for clarifyig this!
Plz answer if you know : Would it be possible that because of low concentration (0.01), the enzyme does not use ddNtp in all probable part to stop replication and as a result we miss one fragment possibility?
how many single-stranded DNA molecules are we putting into those beakers? and.. what if we put too much DNA strand molecules and the ddNTP and dNTP have been used up ( causing repeated base-pairings of the same mass) before the template strand can be sequenced completely?
you just saved my molecular biology exam thanks :)
juan david Andrade lol good luck! let me know how it turns out
hey !! well I passed the exam!! thanks a lot, you a great scientist/teacher :)
thank you so much your lectures are greattttttttttttt
THANK YOU
0:20 how do you know which is the 5 and 3 prime end ? ( without knowing the DNA template ) Is it because you know which end is which from the primer?
If you're using a 100:1 ratio of ddNTPs, how will you sequence the part of the DNA which are longer (travalled the least), the region before the ddNTP?
amazing sir brilliant
THANKS A MILLION TIME 🙏🏻
What happens if we get a full length molecule in the gel electrophoresis...let say if no ddATP was pick to make the complementary DNA molecule in beaker no.1 ...then we obtain a full length molecule plus the 3 shorter molecules. Is this full length molecule runs into the gel too? If so what happens for the reading of the gel electrophoresis?
Sort of like creating 4 equations [using Sanger's method] for 4 unknowns [GACT]
Fascinating
My question is: when the two strand of DNA molecule are separated, how do we do to make sure that in the beaker, we will have only one of the two strand in this beaker. How can we be sure that there is no contamination with the other strand that we do not want?
Thank you so much
Excellent
Sir, I have a question that came in my exam and I didn’t know how to solve it he asked us if we placed a double strand while using sangers dna sequencing what will happen I couldn’t figure it out can u answer me please
Typically, how long a string of DNA can you sequence by this method? Did the scientists define the human genome by this method?
Hi, I don't understand how you got 3 fragments with ddATP, when the DNA Polymerase adds ddATP the synthesis stop isnt it? so how the next fragments are obtained? I would appreciate your help!!!
Thank you
***** thanks! its clear now.
fascinating
Sir u send me the other two methad maxam gilbert and automated dna sequencing plz sir ur the best lacturar
amazing! thank you so much!
❤ lifesaver. Tysm
amazing
Thanks a lot!
Great❤️ thank you sir
Thank you so much :)
How are these fragmentary results assembled into a complete genome?
I am from India sir and your lectures are truly helpful for me and i suggest others also to watch your lectures as u make the concept very clear and understandable with out any complexity. Each and every topic was very well explained and when ever i travel and don't take books along myself i prefer your lectures. I am very thankful of u sir, by seeing your lectures at my room it seems that i am attending lecture. Not before exams but also during my free time i watch your lectures to clear my concept and there is revision of basics also. Thank u so mch.
Regards: Shantanu tomar
perfect! thank you so much
Well explained
Legend!
Ty❤
My hero
We need to revise the lecture back
Thankssssssss
LEGEND