omg never believe I came back to this channel again when finding methods to solve my research！ I truly hope you are having a peace&healthy life in India, no matter how hardship the covid is for our generation. if I had a chance to visit India someday after graduation next year,I am really looking forward to visiting India & maybe find a chance to have a tea with Teacher Shomu! The honor would be mine:)
Vector construction is okay with terminal deoxyribonucletidy transferase enzyme along with dTTP. but to attach the Adenosine in 3 prime end will be of no use, because in vector construction the terminal transferase enxymes attaches nucletides at 3 prime end also. to avoid this we can add poly T tail in insert dna prior to PCR by using terminal transferase enzyme,then design primer particular adenosine primer sequence complementary to poly T tail of insert dna. After PCR ,nascent dna with poly A tail at 5 prime end will be produced. which can be inserted in the vector .
But what is the reason as to why we do not need a restriction digestion for the vector? And after binding is when we could do the PCR? Instead of adding A at the 3' end via Taq DNA polymerase and T to the 5' end via TDT can't we use a RE and produce sticky ends so that they can ligate via DNA ligase?
Doc Hana Because 3' end will form phosphodiester bond with 5' end of Thymidine. Now 3' end of Thymidine can form phosphodiester bond with 5' end of the DNA with the A sticky end, resulting in insertion.
Thank you for your explanation and effort. My question is, briefly, why is it a disadvantage that it is not directional? In other words, what is the importance of directionality in such methods?
Thank you for this very interesting and clear video. I have just a question: in order to discover the orientation of my insert in the vector, I can perform a PCR with M13 fw and rev and T3-T7 primers...but I do not really understand how they work. Could someone help me?
can some one explain please how if ithe insert can go into the vector in both orientations. how it will affect the product of the genes? what happen in both cases what is the diffrences
the T-vector will usually have a promoter, or simply an origin of transcription upstream of the inserted target DNA. If the DNA is inserted correctly, the gene can be expressed. if the DNA is inserted the "wrong way around" so to speak, it would be like reading a book from right to left. The expressed protein will not be active or in most cases the gene wil not be expressed at all
usually to subvert this issue, they have a toxic gene sequence in the antisense ORFs that way they ensure that only the correct orientation of the gene-containing plasmids survive.
Wish I could go to your college classes instead of my “elite” university that doesn’t bother to employ dedicated teachers only researchers who don’t give rats arse! 😭😭
omg never believe I came back to this channel again when finding methods to solve my research！
I truly hope you are having a peace&healthy life in India, no matter how hardship the covid is for our generation.
if I had a chance to visit India someday after graduation next year,I am really looking forward to visiting India & maybe find a chance to have a tea with Teacher Shomu! The honor would be mine:)
Thank you so much for appreciating my efforts. Glad to hear that you're getting benefit from my lectures
your videos got so much better compared to the ones from 4 years ago, keep up the good work and thank you for posting this
im watching this in 2024 and its still one of the best explained lecture and even his website with all articles 😍
One of the best teacher in my view. U teach in such lucid way. Your videos helped me a lottttttt to perform well during my MSc exam.
Thank you so much for appreciating my efforts
I believe TdT adds nucleotides to the 3' end. Great video btw.
yes same doubt
It the best bio channel I ever seen
Vector construction is okay with terminal deoxyribonucletidy transferase enzyme along with dTTP.
but to attach the Adenosine in 3 prime end will be of no use, because in vector construction the terminal transferase enxymes attaches nucletides at 3 prime end also.
to avoid this we can add poly T tail in insert dna prior to PCR by using terminal transferase enzyme,then design primer particular adenosine primer sequence complementary to poly T tail of insert dna. After PCR ,nascent dna with poly A tail at 5 prime end will be produced. which can be inserted in the vector .
The terminal transferase adds Thymine nucleotide to the 3' end of the linearised vector.. great video though!
Hai.. i am comfused.. why not 5' end? I am newbie in genetic
How does Taq polymerase add a A to the end of the template? WOuldn't it just be easier to put the A in the primers?
How is the frame taken care of?
But what is the reason as to why we do not need a restriction digestion for the vector? And after binding is when we could do the PCR? Instead of adding A at the 3' end via Taq DNA polymerase and T to the 5' end via TDT can't we use a RE and produce sticky ends so that they can ligate via DNA ligase?
Why we not use GT nucleotides in target DNA and T vector
does not TdT add nt to the free 3'ends for each strand of DNA?
TdT adds ddTTPs to 3'-OH not 5'end , both will produce 3' overhang
Kindly review your video,, Tdt transfer nucleotide to 3' end not to 5' end.
Yes!!!that's why I can't figure out how the PCR cloned into the vector. it will become 3' end to 3'end ! quite weird !!!
Vikas K Singh
how they could binds with each other if the bothe ended by t at 3'prime end??
Doc Hana Because 3' end will form phosphodiester bond with 5' end of Thymidine. Now 3' end of Thymidine can form phosphodiester bond with 5' end of the DNA with the A sticky end, resulting in insertion.
what i dont understand is how the taq polymerase can add an A in the end of PCR fragment? without a template?
Taq pol has terminal transferase activity of adding single A to 3' end
This video was very helpful. keep up the good work
Thank you
This is such a helpful video! Thank you so much!
You're welcome. Glad to hear that you're getting benefit from my lectures
why we shouldn't use G/C cloning like T/A cloning?
Can u Plzz make vedio on infusion cloning
Okay
LOVE YOU SHOMU SAVED MY LIFE
You're welcome
Your videos help a lot...
+Esther Sharon nice to know that
thanku for such such kind of informative videos
Glad to hear that you're getting benefit from my lectures
Thank you for your explanation and effort. My question is, briefly, why is it a disadvantage that it is not directional? In other words, what is the importance of directionality in such methods?
Very informative Sir!
Thank you for this very interesting and clear video. I have just a question: in order to discover the orientation of my insert in the vector, I can perform a PCR with M13 fw and rev and T3-T7 primers...but I do not really understand how they work. Could someone help me?
One question please in GC cloning do we require DNA ligase, because there are three bond in GC ?
can some one explain please how if ithe insert can go into the vector in both orientations. how it will affect the product of the genes?
what happen in both cases what is the diffrences
the T-vector will usually have a promoter, or simply an origin of transcription upstream of the inserted target DNA. If the DNA is inserted correctly, the gene can be expressed. if the DNA is inserted the "wrong way around" so to speak, it would be like reading a book from right to left. The expressed protein will not be active or in most cases the gene wil not be expressed at all
usually to subvert this issue, they have a toxic gene sequence in the antisense ORFs that way they ensure that only the correct orientation of the gene-containing plasmids survive.
This Video was helpful Sir
You're welcome
perfect explanation Sir
Thank you 💕
You're welcome
I have q question. Why we do TA cloning when we already did the pcr for making of multiple copies.??
I think after making multiple copies by PCR we need to know the sequence of that product. So for sequencing purpose we perform cloning.
Why it only add one adenin?, i mean like exactly 1, why not 2 or more (poly a)
Fantastic work! Thank you!
You're welcome
who is hollering in the background :|
Awesome video, very useful. Thanks m8
You're welcome. Glad to hear that you're getting benefit from my lectures
you are the best!!!!!!!!!!!!!!!!!!!!!!!!!!
Thank you
Thank you very much sir
You're welcome. Glad to hear that you're getting benefit from my lectures
You should team up with Khan Academy
+Brendan Law thank you.
Shomu's Biology you are very good and so easly explaination
Dhanyavad
Vector has T overhang at 3' end
Sir...can u plz help me solving my biotechnology assignment
please increase the recording volume. :)
Thank you!
You're welcome
Thank you
Thank you so much! Your videos are awesome!
+Nicole Senff thank you. Glad you liked my lectures
+Nicole Senff thank you. Glad you liked my lectures
Thanx
You're welcome
Thank you sir, it's really helpful .
You're welcome. Glad to hear that you're getting benefit from my lectures
Make a video explaining difference between blunt end and sticky end cloning
Okay
Wish I could go to your college classes instead of my “elite” university that doesn’t bother to employ dedicated teachers only researchers who don’t give rats arse! 😭😭
What would I say
Pls increase you voice 😣
Ok
i love yoou
Thank you.
Thanks a lot! Your videos are amazing!
thank you
You're welcome