Thank you so much! I'm involved in a project where they asked me to design primers for the first time and your detailed explanation saved me, thank you again for your work and for sharing your experience with us!
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.
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
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
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
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
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.
@@akosiletaiwo9927 Pick the target sequence and paste on the NCBI database, they will generate numbers of primers. From there pick the primer which seems to be suitable for you. Consider the length, GC%, and annealing temperature
@@akosiletaiwo9927 The GC content should be approximately 40-60%, with an annealing temperature of 60 ± 5°C. The primer length should be between 18 and 25 nucleotides. For specificity, the number of mismatches should be less than 3.
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?
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?
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%)
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 so much! I'm involved in a project where they asked me to design primers for the first time and your detailed explanation saved me, thank you again for your work and for sharing your experience with us!
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
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
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
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.
Hi there, I am sure you have firgured this out, how did you get it please
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.
I am so happy I found you!!
Very informative. I am a beginner and this is what I was looking for. Thanks.
GOOD LECTURE 👍GOD BLESS YOU TEACHER 😊
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
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
Really good explanation!!
Helped a lot.
Great video, thank you! Buto use say from a DNA sequence, but the sequence you use as an example is an mRNA sequence
I could not find the description table I need the websites, please
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
Thanks! This is a good video!
Thank you so much your perfect lesson of primer design!!
This is exactly what i needed, thank you so much
Maybe add these links for sites in the description below of the video?
And what will be the amplicon length for the amplified product after PCR?
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!
How the forward primer would bind to template strand? because both have the same sequence
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
Thanks, that was helpful
Your vids are concise and very simple to understand
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
Well understood. Thank-you
The link isnt working for me? Has anyone else had success and can post the link?
Thank you very much!
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
I don't understand what the "optimized" primers are. What are they optimized FOR?
This is very helpful! Thank youu!
Thank you , if I have primer F : 66 melting temperature
and the primer R: 68
Which suitable annealing temp for this gene ?
What about the quality of the primers are they any good in terms of the Tm and self complementary?
Where are the links ????
THANK YOU LIFE SAVER
Why use complementary sequence for RP but not for FP..??
very helpful, thank you !
but how would you check for dimer formation ?
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 there, I'm sure you have figured this out now, please how did you do this?
@@akosiletaiwo9927 Pick the target sequence and paste on the NCBI database, they will generate numbers of primers. From there pick the primer which seems to be suitable for you. Consider the length, GC%, and annealing temperature
@@akosiletaiwo9927 The GC content should be approximately 40-60%, with an annealing temperature of 60 ± 5°C. The primer length should be between 18 and 25 nucleotides. For specificity, the number of mismatches should be less than 3.
@@सविन-झ4झ Thank you
Thank you for this awesome vidoe.
Can you please tell.me how can we check the orientation of DNA sequence. Please help
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
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?
I just did my question paper with your explanation if I don't get marks I m going to hunt you down 😂😛
Where you brought these genes first?
Why we need partial sequences whlie designing of primer?
In the video why forward and reverse primer has same 5 prime to 3 prime direction
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?
Sir reverse primer is 3 to 5 direction or 5 to 3 direction please tell me the answer?
Thank you!
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?
But how will you check if there are off-target amplification?
Can we make the primer for whole gene for qPCR
How about a link to those websites? Wouldn't that be useful...
Thanks a lot❤
Hi, what app did you use to make the video - with the coloured pens on the right? What tool is this, please?
Does this apply for RT-PCR as well?
Template is an RNA virus.
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?
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
perfect
But amplification of large gene with only one primer isn't trustworthy or may have incorrect base sequence
Why cant we get people like this in universitys?
how to design probe
When they say "it lacks the primer for reverse transcriptase" ... uhm, it can make its own!
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 ...