It has to do with the nature of the molecule itself and the way in which the carbon bonds are numbered. The only way to give a sense of direction is to do it this way. Basically its all about organic chemistry. a detailed explanation can be achieved via wikipedia on the page for Directionality (molecular biology). It gives you a nice visual as well that really helps to understand
hey.. thnx for all of ur vid.. really helps for newbies :D just wanna ask somethn' this is my first time to design primer and i will be dealing for cloning; i was given a vector that hv 2 mcs. do i need to use differ re for both reverse and forward? and what about the frame shift thing. dont really understand..
Thanks for featuring SeqBuilder! To learn more about designing primers in DNASTAR software, this video may also be useful. DNASTAR - Primer Design Overview
hey. thank ya for the vedio. however, i have a problem hope ya can help. how then do i look for my Tm since its important and specific??? also, the GC content is very important, what if it is less than %o%, what are ways to make them a bit better??
Great video. I am just confused why the forward primer was complimentary to the bottom strand and the reserve primer was complimentary to the top strand? I also would appreciate it if you can explain it to me how I would design a primer for a cDNA. Thanks!
+Yakin Jaleta Thank you for checking out my video. As for your questions - because that's how DNA polymerase works, always synthesizing from 5' to 3' ends. In that case the forward primer must bind to the template strand while the reverse primer must bind to the coding strand. Designing primers for cDNA is no different from the standard process - not quite sure exactly what you're asking.
I'm sorry, but I don't see the problem. I looked at the forward and reverse primers in the video, written from the 5' to 3' direction, and they are not complementary to one another. I mean I guess there are maybe a few bases here and there that are complementary, but that's unavoidable when designing primers. Usually, there will always be some degree of dimer formation just from the PCR process and it also depends on your specific cycling conditions. I hope this helps! And thanks for checking out my video!
The primers are not complementary to each other at all. Think of it this way. You have two DNA strands, the leading strand in the forward 5 to 3 prime end and the lagging strand in the backward 5 to 3 prime end. These strands are complementary to each other. Now break the bonds between these two strands and make them single stranded. The primer that binds to the lagging strand will be complementary to it, so it will be an exact replica of the leading strand.... this is called the forward or sense primer..... the other primer or the rather the primer that binds to the leading strand will be a complement of the strand in the reverse. This is called the reverse or antisense primer.... So when you think of it.... We have two primers... the forward primer being the exact replica of the leading strand and the reverse or antisense primer being a "reverse complement" of the the lagging strand.
Hey, thank you for the video, it's been very helpful! However i've got a little curiosity: once you have your primer designed in software, how do you get it? Do you synthesize it in a lab or do you extract it from a living cell?
I thought the primers needed to have some spaces about 18-25 before the desire sequence in order to get the starting codons and you would lose some while processing it....
Very good video, nice man I love it
Thank you, you've refreshed my memory, this video was very usefull to me!
hello. i think it would be helpful if you also mention what kind of software you use to get your DNA.
Cool video. Just got into PCR today in my Bioinformatics class. This helped, will check into your other videos.
Thank you. I'm glad you found it helpful.
Hello thanks for the video .. can i know what the program name that u used ?
It has to do with the nature of the molecule itself and the way in which the carbon bonds are numbered. The only way to give a sense of direction is to do it this way. Basically its all about organic chemistry. a detailed explanation can be achieved via wikipedia on the page for Directionality (molecular biology). It gives you a nice visual as well that really helps to understand
hey.. thnx for all of ur vid.. really helps for newbies :D
just wanna ask somethn' this is my first time to design primer and i will be dealing for cloning; i was given a vector that hv 2 mcs. do i need to use differ re for both reverse and forward? and what about the frame shift thing. dont really understand..
Wish this had more detail because many times there are things to look out for when designing primers.
Thanks for featuring SeqBuilder! To learn more about designing primers in DNASTAR software, this video may also be useful.
DNASTAR - Primer Design Overview
Thank u this is very helpful! But u explain why reverse perimeter is needed?
So helpful! Thank you!!
Do primers have uracil substituted for thymine?
hey. thank ya for the vedio. however, i have a problem hope ya can help. how then do i look for my Tm since its important and specific??? also, the GC content is very important, what if it is less than %o%, what are ways to make them a bit better??
I was wondering... What would happen if the two primers were on the same strands ?? Would there be any PCR's product or nothing ?
Identify the first 21 bp sequence of the sense strand of pGEX-6p1 when it’s linearised?
Thank a bunch! This is very helpful
Great video. I am just confused why the forward primer was complimentary to the bottom strand and the reserve primer was complimentary to the top strand? I also would appreciate it if you can explain it to me how I would design a primer for a cDNA. Thanks!
+Yakin Jaleta Thank you for checking out my video. As for your questions - because that's how DNA polymerase works, always synthesizing from 5' to 3' ends. In that case the forward primer must bind to the template strand while the reverse primer must bind to the coding strand. Designing primers for cDNA is no different from the standard process - not quite sure exactly what you're asking.
+TheScyther88 I figured it out. Actually, thanks for your videos I got 100% on my take home exam!
+Yakin Jaleta Good job! :)
I don't understand why the primers are complimentary to each other? Won't it form primer dimers galore?
I'm sorry, but I don't see the problem. I looked at the forward and reverse primers in the video, written from the 5' to 3' direction, and they are not complementary to one another. I mean I guess there are maybe a few bases here and there that are complementary, but that's unavoidable when designing primers. Usually, there will always be some degree of dimer formation just from the PCR process and it also depends on your specific cycling conditions. I hope this helps! And thanks for checking out my video!
The primers are not complementary to each other at all. Think of it this way. You have two DNA strands, the leading strand in the forward 5 to 3 prime end and the lagging strand in the backward 5 to 3 prime end. These strands are complementary to each other. Now break the bonds between these two strands and make them single stranded. The primer that binds to the lagging strand will be complementary to it, so it will be an exact replica of the leading strand.... this is called the forward or sense primer..... the other primer or the rather the primer that binds to the leading strand will be a complement of the strand in the reverse. This is called the reverse or antisense primer.... So when you think of it.... We have two primers... the forward primer being the exact replica of the leading strand and the reverse or antisense primer being a "reverse complement" of the the lagging strand.
It is my question too!
No problem! Glad it helped you out.
Short and simple description
Thanks for watching everyone! I will be back soon with more videos. Stay tuned.
Hey, thank you for the video, it's been very helpful! However i've got a little curiosity: once you have your primer designed in software, how do you get it? Do you synthesize it in a lab or do you extract it from a living cell?
you order it from a company that makes primers. (thermofisher, IDT)
newbie question: why do they call it 5' and 3'?
how to Designing a set of PCR primers for amplification of the linearised pGEX-6p1? using ApE
THANK YOU! So helpful!
How do u make em I n lab?
thank you soooooooo much ! saved my life !
What program is that?
Nora SD It's SeqBuilder, part of the Lasergene suite. I have a tutorial about it as well.
great video
Thank you.
I thought the primers needed to have some spaces about 18-25 before the desire sequence in order to get the starting codons and you would lose some while processing it....
Please, send me the software aplicate to tecnics. Tks!
Thank you sir!
There's no software to get DNA sequences. You can find different sequences on NCBI, commercial websites, and place of work/research.
No I meant the software that you use to open your sequence
Dr. Scyther!
Thanks
SeqBuilder. I have a video up about that already.
Ops my bad. You're right
No problem, I'm glad it's clarified :)
No, you do not substitute U for T.
If this video got you a masters degree, I want to know where it is and why I didn't go. This is an undergraduate lab procedure for most institutions.
which is the program that you are using?
SeqBuilder, part of the Lasergene suite.