Many thanks for video. 1. Primers you designed, how to get to know the annealing temperature of both primers and GC . 2. What is difference between full length primers and short length primers means why we do. For example one gene express in full length primer in semi-qPCR but in short length designed primer this isn't expressing.
its very helping to me.. i was looking for target gene forward and reverse primers form.published literature but it was lacking number of nucleotides and qPCR protocol for ea ch target gene.. the published literature only contain gene accession code where the origin gene sequenceing section can be obtained .. bu using available information in literature i could be able to design primer by learning this tutorial.. Thank u for sharing this Knowledge
Because DNA polymerase adds chain 5' to 3' so first replication starts with reverse primer. If we want to copy newly formed chain it's complementary to it (actually it's same as original code) Just imagine how PCR works and you will understand
Thank you so much for your video. I am wondering how we check the accuracy of the design primer. Is there any website like you mentioned in the video? I wanna see if my design has appropriate Tm, and GC content and no secondary branching.
Hi, in the second method the forward primer is same as template sequence, it should be complementary if it has to go and bind to template strand, how it's gonna work?
@@bhagatsingh6345 im sorry, but could you please elaborate more ? i couldnt seem to understand how the forward primer will bind even if its read the other way
Great, but did not explain why the Primer -Blast did not work and you had to use another tool? also why did you not specifiy amplification ranges in the primer blast?
Hey there! Thanks for the video. What if the forward primer as described in the video contains GC and AT rich regions which are not desired? Or if they have suboptimal melting temperatures? Would one then have to search for other 20mers upstream and downstream of the gene to use instead of the start and stop regions as starting points for the 20mers? And how would we do that? Is there a tool for that also? Kind regards
The start-point and endpoint of a desired DNA-sequence does not provide necessarily a suitable set of primers. In most cases, it does not. (Selfdimers, wrong amount of Cytosines en Guanines. CG-amount should be around 60%)
we need to add the restriction site before the forward primer and reverse primer and we also need to add the stuffner nucleotides before the restriction site but do you have nay idea how many base pair we can add before the restriction site?
Your explanation for the forward primer makes no sense tbh. How is that primer going to attach to the first 20 base pair sequences of that strand when it's the exact same and isn't a reverse compliment?
THANK YOU! I have a project where i need to design a primer and your video genuinely saved me and explained it suuuper well too!
Same here. Thank you so much.
FINALLY!. Exactly what I was looking for! Thank you!
...me too
Many thanks for video. 1. Primers you designed, how to get to know the annealing temperature of both primers and GC .
2. What is difference between full length primers and short length primers means why we do. For example one gene express in full length primer in semi-qPCR but in short length designed primer this isn't expressing.
Thank you! Great explaination! I have to do this for my homework and this is the first video that really helps! Thanks
Thank you!
When you finished designing the primer how can we now use it for our PCR?
The video is easy to comprehend and implement in primer design. Thank you for a job well done
From here you would have to deterimine the Tm for each and hope that they are close to each other for the PCR to work.
This is the best explanation I have come across!! Thanks
Wow! this is just the answer to the questions I have been trying to ask. THank YOu
Very informative. I am a beginner and this is what I was looking for. Thanks.
its very helping to me.. i was looking for target gene forward and reverse primers form.published literature but it was lacking number of nucleotides and qPCR protocol for ea ch target gene.. the published literature only contain gene accession code where the origin gene sequenceing section can be obtained .. bu using available information in literature i could be able to design primer by learning this tutorial.. Thank u for sharing this Knowledge
I am so happy I found you!!
Thanks! This is a good video!
Nice explanation, you helped me alot with my homework. Thanks very much!
No problem!
@@CatalystUniversity can you hlp me
It is very useful for me , thank you so much
GOOD LECTURE 👍GOD BLESS YOU TEACHER 😊
Thank you so much, perfectly to the point. would you also please make a video on how to design a primer for SNP genotyping? or is it the same?!
Hi, did you find a video on this? Im interested in designing primers for SNP detection and I havent found any good information on this.
@@karinacaetano4016 no unfortunately, I think this kind of information is only given in paid courses and for free!
Maybe add these links for sites in the description below of the video?
Why Forward primer sequence remains same as complementary strand ?
Because DNA polymerase adds chain 5' to 3' so first replication starts with reverse primer. If we want to copy newly formed chain it's complementary to it (actually it's same as original code)
Just imagine how PCR works and you will understand
Your vids are concise and very simple to understand
Thank you so much your perfect lesson of primer design!!
I could not find the description table I need the websites, please
Thanks, that was helpful
Your reverse primer doesn't end with a C or a G. Would it work better if it was one base longer to get a better "3' clamp"?
I think it’s better to have a GC end based on my research
Thank you so much for your video. I am wondering how we check the accuracy of the design primer. Is there any website like you mentioned in the video? I wanna see if my design has appropriate Tm, and GC content and no secondary branching.
Really good explanation!!
Helped a lot.
This is exactly what i needed, thank you so much
Well understood. Thank-you
The link isnt working for me? Has anyone else had success and can post the link?
I just did my question paper with your explanation if I don't get marks I m going to hunt you down 😂😛
Thank you very much!
THANK YOU LIFE SAVER
Where are the links ????
Hi, in the second method the forward primer is same as template sequence, it should be complementary if it has to go and bind to template strand, how it's gonna work?
primer also recognised to 3' prime end and DNA Pol 5' to 3'.
@@bhagatsingh6345 im sorry, but could you please elaborate more ? i couldnt seem to understand how the forward primer will bind even if its read the other way
Exactly my question
Why use complementary sequence for RP but not for FP..??
Great, but did not explain why the Primer -Blast did not work and you had to use another tool? also why did you not specifiy amplification ranges in the primer blast?
Can you put out some practice questions for us? Thank you
This is very helpful! Thank youu!
very helpful, thank you !
How about a link to those websites? Wouldn't that be useful...
Hey there! Thanks for the video. What if the forward primer as described in the video contains GC and AT rich regions which are not desired? Or if they have suboptimal melting temperatures? Would one then have to search for other 20mers upstream and downstream of the gene to use instead of the start and stop regions as starting points for the 20mers? And how would we do that? Is there a tool for that also?
Kind regards
for the reverse primer, why did you not change the 5 prime to 3 prime and 3 prime to 5 prime.i actually thought you forgot to do that.
It is a reverse complement, so it is running from 5 to 3 on the complementary strand
Thanks 😊
You're welcome!
Thank you great video. But does your fp need reverse complement as well? Thxxx
And why u use 40mer so big? You need high accuracy?
Thank you for this awesome vidoe.
Can you please tell.me how can we check the orientation of DNA sequence. Please help
Sir I like your video I want to confirm that reverse primer is 3 to 5 direction or 5 to 3 direction please tell me?
Thank you!
What about the quality of the primers are they any good in terms of the Tm and self complementary?
I don't understand what the "optimized" primers are. What are they optimized FOR?
The start-point and endpoint of a desired DNA-sequence does not provide necessarily a suitable set of primers. In most cases, it does not. (Selfdimers, wrong amount of Cytosines en Guanines. CG-amount should be around 60%)
How do you mitigate against this?
Thank you , if I have primer F : 66 melting temperature
and the primer R: 68
Which suitable annealing temp for this gene ?
How the forward primer would bind to template strand? because both have the same sequence
In the video why forward and reverse primer has same 5 prime to 3 prime direction
but how would you check for dimer formation ?
Thanks a lot❤
But how will you check if there are off-target amplification?
Sir reverse primer is 3 to 5 direction or 5 to 3 direction please tell me the answer?
Hi, what app did you use to make the video - with the coloured pens on the right? What tool is this, please?
Why we need partial sequences whlie designing of primer?
we need to add the restriction site before the forward primer and reverse primer and we also need to add the stuffner nucleotides before the restriction site but do you have nay idea how many base pair we can add before the restriction site?
I need to do a similar thing, just wondering how you worked this out?
Why cant we get people like this in universitys?
Where you brought these genes first?
Does this apply for RT-PCR as well?
Template is an RNA virus.
But amplification of large gene with only one primer isn't trustworthy or may have incorrect base sequence
Can we make the primer for whole gene for qPCR
perfect
When they say "it lacks the primer for reverse transcriptase" ... uhm, it can make its own!
how to design probe
Hey can I send you a message ?
what
Your explanation for the forward primer makes no sense tbh. How is that primer going to attach to the first 20 base pair sequences of that strand when it's the exact same and isn't a reverse compliment?
Sir I wanna a contact you I need you help ...