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Primer Design for PCR

1915 ratings | 372466 views
In this lecture, I explain how to design working primers for use in PCR. If you are unfamiliar with PCR, watch the following video: http://www.youtube.com/watch?v=2KoLnIwoZKU. Created by:Tyler Maxfield
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Text Comments (99)
Alex Cracker (29 days ago)
At 11:50 you say :"This low temperature annealing G and C means that when the temperature is increased in PCR this primer physically can not bind...." It is not correct. The primer with higher GC-content required higher melting temperature.
Ghost Medic (1 month ago)
This video would have been significantly more helpful if we could have seen everything on the screen you were pointing to. On our end, your pointing and talking about something that is a couple inches from or visible screen. Thanks anyways though.
Guzinato r (2 months ago)
play it on 1.5 speed, better to follow
Linus Bao (3 months ago)
thank you so much, very helpful
Beauty P. (3 months ago)
Very helpful!
Marc Kramer (5 months ago)
you are a true hero
Stephen Rose (6 months ago)
I'm confused, if the DNA polymerase binds to the 3' end and goes in the three prime direction, there's no where to go, its already at the end?
Rameen Mohsin (6 months ago)
why it is necessary to have G or C on the end of our sequence?
Linus Bao (3 months ago)
more hydrogen bonds between g and c vs a and t
Johana Jaramillo Guzmán (6 months ago)
thank you soo much!
Desy Samsung (7 months ago)
Great job!
Sally Cha (7 months ago)
Best explanation! Thank you so much!
Sunita Kumari (8 months ago)
sir, both primer are complementary to each other like dsDNA?
spagetti001 (8 months ago)
wow you're not indian, how can this be
曾慕楚 (9 months ago)
very helpful! thx very much
Phagy Auto (10 months ago)
Yara Edor (1 year ago)
tatie zaty (1 year ago)
Hi, it is about SSR marker. how may I know if the ssr has most fixation and similar number of genetic fingerprint?
Haven Kerley (1 year ago)
thank you
Kasia Janiszewska (1 year ago)
Thank you for the video!! I learned something ;)
MJ Resina (1 year ago)
So useful
N T (1 year ago)
Great , thank you very much
vishwambar navale (1 year ago)
It's really helpful
Sophie G (1 year ago)
stupid question: when does the DNA pol. stop copying the strand? How many bp from the primer?
Abel Babel (1 year ago)
Polymerase stops wherever the template ends
Sophie G (1 year ago)
Wait wait wait... just where the two primers end, so you get dsDNA between the primers?
Nadera Noori (1 year ago)
thank you so much, actually you have save my many hours of studying, to get to learn how to design a primer.. keep it up
Hamed Hosseini (1 year ago)
https://www.youtube.com/watch?v=Nt2DQnpkLBE Give my video a view i've gone through application problem.
Marla Miranda (1 year ago)
Very helpfulll, thank you so much !!!
Thank you!!! I understood it perfectly. And guys, If you have SOME idea of basic genetics you wont need the view of the whole screen... Greetings from argentina!
Neel Parmar (1 year ago)
Fantastic vid, thanks so much
Gram T (1 year ago)
what are sticky ends for?
frank addeo (1 year ago)
thanks a lot!
Wow, thanks amazing video
MrMetalHead1100 (1 year ago)
Thanks bro. That was everything I needed!
Ryan Wonderlin (2 years ago)
Do you need to include your start codon in your forward primer?
What do you need a start or a stop codon for in a PCR?
Ryan Wonderlin (2 years ago)
What about the stop codon in the reverse?
Aregawi Ataklti (2 years ago)
please could you help me on primer design
Maryam Bukhari (2 years ago)
Very helpful. Thank You
Opa Lee (2 years ago)
Why do you have to have 20/21 bases for the forward primer, must you? Or is it just desirable?
Waterloo Chavez (1 year ago)
also it would be less likely for there an equal sequence if the length is greater than 17-18 bp
Ice Lysis (2 years ago)
Found this on the internet: Primer Length: It is generally accepted that the optimal length of PCR primers is 18-22 bp. This length is long enough for adequate specificity and short enough for primers to bind easily to the template at the annealing temperature. Hope that helps!
Umoss Col (2 years ago)
i stil don't understand the fact that we have to reverse the primer, I mean it would still come out the same?
Jake Qiu (2 years ago)
It's simply when you are buying and ordering your primers from somewhere. Because the structure will be different if you go from 3' to 5' and 5' to 3' and if you ordered it as 3' to 5', you'll be getting a 5' to 3' primer from the vendor and it won't work as you imagined it to do.
krabatenn (2 years ago)
This may be a silly question... What determines when the DNA polymerase will stop adding nucleotides to the single strands?
Nicolas Diaz (2 years ago)
that's not correct, RNA Pol stops in the stop codon, the stop of DNA Pol is determined by different factors, like it's processivity, a DNA pol can amplify up to 30kb under the right conditions.
KnackeredSchizo (2 years ago)
When a DNA pol encounters a STOP codon it disengages from the strand, so that will stop the synthesis.
賴TERRYLAITW (2 years ago)
very useful and informative. Great job there
Nour Ghaddar (2 years ago)
Thank you amazing video !
Joe B (2 years ago)
Hi, I am unclear about the "reverse complement" explanation at 9:05. You say if we send in the sequence as is that we would get something thats 5' -> 3' in the wrong direction. Why is that? the way you wrote the sequence has the 5-3' orientation which looks correct. Please explain. Thank you .
Shahed and Science (2 years ago)
+Joseph Beyene you welcome Joseph. I created a video on PCR on my YouTube channel three days ago. Kindly check it out and let me know your comments. Thank you very much.
Joe B (2 years ago)
Very clear. Thank you
Shahed and Science (2 years ago)
Hi Joseph. When we send the primer sequence for a company, the direction has to be in the 5' to 3' (we read the sequence direction from left to right) direction for both, the reverse primer and the forward primer, to prevent confusion. the company will never ask you if you want your primers to be in the in the 5' to 3' direction or in opposite direction, they will just design it, and the default direction followed by all companies is 5' to 3' direction. if you look at the minute 9:05, the direction of the designed forward primer is already in the 5' to 3' direction. but in the case of the reverse primer, the above sequence, the direction is 3' to 5' , if you send this sequence to the company in the 3' to 5' direction, they will design it for you in the 5' to 3' direction because it is the default direction for them, therefore you will end up having the wrong sequence. you have to do something called reverse complement, in which you rewrite your sequence in the 5' to 3' direction, and then send it to the company. let me know if you have further question.
Nataly GG (2 years ago)
Thank you for the precise explanation! You really helped me
maddy (3 years ago)
Sudhir Sawarkar (3 years ago)
Simple and clear explanation ... thank you
Dongyang Zhang (3 years ago)
Haifa Mansour (3 years ago)
very simple and clear explanation.. Thanks
ertosns (3 years ago)
+Haifa Mansour that is a good website
Haifa Mansour (3 years ago)
write ''ss'' before ''youtube'' in the link.
ertosns (3 years ago)
+Jyoti Mishra https://www.google.com.eg/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=youtue+downloader+online
Jyoti Mishra (3 years ago)
bt hw can i download it
Benji Price (3 years ago)
thatguywhodoesstuff (1 year ago)
Benji Price The tools are boring, but it's applications in modern biology have been quite the opposite.
Running99fly (3 years ago)
Aaron Ledray (3 years ago)
Thanks! Great refresher.
Danielle Belize (3 years ago)
Thanks, it's been a while since I had anything to do with primer design. It was fun to relive it.
Yimu Yang (3 years ago)
I m more addicted to the gentle voice than the helpful video lol thanks!
Viviane Girardin (3 years ago)
Very easy to follow and well explained, thank you!
Alison Lee (3 years ago)
Thank you so much for this!! I looked everywhere on the internet and for some reason, the way you worded it specifically and drew everything out just made so much sense to me!! Thank you!!
rawrafied (4 years ago)
Thank you for this. It was very helpful! ^_^
ramesh tiwari (4 years ago)
at 9:00 you meant to write reverse, but instead, you wrote forward. 
DogeterPhil (3 years ago)
+ramesh tiwari I think he might have done that deliberately, because if you write the sequence that way without noting down the 3' and 5' ends, it's identical to the forward primer, as he explains
Shannon Wu (4 years ago)
too slow-paced
Joanna Thomas (4 years ago)
Shame that the bottom of the screen is cut off
J AI (4 years ago)
i fucking hate molecular biology
mehdi ched (2 years ago)
i totally agree with you ! this is the bad thing about a 'degree' and 'diploma' they just want you learn a ton of useless boring long courses you don't need'em, just to pass an exam, and Molecular Biology it's just fascinating beautifull and powerfull..
J AI (2 years ago)
I don't hate it i just hate having to learn something in pressure for an exam just to finish a course and move into the next course and not truly understanding the previous course because molecular biology truly is fascinating. I want to learn to understand and not just to finish a course. However I'm finished with my bachelors now and can read in peace and learn freely without the pressure of having to get passed on a crucial course.
mehdi ched (2 years ago)
Arjun (4 years ago)
Thanks a lot ....Great help
mary grace sedanza (4 years ago)
THANKS i learned many things! can someone help me go about designing a degenerate primer for aquatic species?i would greatly appreciate it
Hira Khan (4 years ago)
Hi! I am having some problem designing my primer. I need to amplify my gene of interest but the forward primer GC% does not go higher than 29%. ATGAAGAACTGCCTAAAAATGAAAAACC. Even if I extend it the Tm becomes too high.  My Tm really needs to be at 60degrees. I would really appreciate if you could help me as I am in a serious pickle!
thatguywhodoesstuff (1 year ago)
Hira Khan how'd it go man?
krishna chaitanya (4 years ago)
Nice explanation, but one suggestion when you compare sequences, try to put the video frame in such a way that both the sequences are seen at a time. 
Angelus Aeternam (4 years ago)
I want to ask, why is 3 prime and 5 prime standard convention? Is there a video where I can check this out?
Ozan Şanlı (4 years ago)
Wonderful work my friend , please keep doing this !  Regards
Sumana Fathima (4 years ago)
thank you!! I was so confused until now. THANK YOU
93lookatmenow (4 years ago)
omg this is so easy than you i was so confused in class damn 
Cayla Naumann (5 years ago)
Great video!  My only comment would be to be a bit more aware of what can be seen on screen at a given time, as sometimes things were a bit cut off at the edges.
Marina ferreira lima (5 years ago)
Nicole Crosby (5 years ago)
good one!thank you)
Joana Reis (5 years ago)
Awesome explanation. Obrigada! 
kevinm (5 years ago)
devilnot6 (5 years ago)
DP lol
very well explained
Lafia Sebastian (5 years ago)
Very good video...
julles81981 (5 years ago)
This is a great video thank you so much
Nazim Medzhidov (5 years ago)
Thank you for this video!!!

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