Primer designing for real time PCR using NCBI Primer Blast

This video explains how to design primers for real time pcr using primer blast.
NCBI's primer blast tool helps in designing primers and also allows to check the specificity of the designed primers.
Various options are available to improve the primer stringency.

[@annavl4937]

Hi, thanks for the video! Another way I know to avoid potenitial genomic DNA amplification is by selecting "Primer must spand the exon-exon junction", so no intron-including DNA would be amplified.

[@krishnaprasad5508]

Thank you very much. It was beautifully explained.

[@Scienceiirwn]

Amazing Video!!! Is there anyway you can do a video on designing a primer to target different variants ? for example i noticed in your example there are many GAPDH variants (V1 -V4), what if you want to target the different variants to see the expression of each one?
I would really appreciate if you can give an example using CD44 which has 10 variants.

[@anuranjanapv4484]

Hello sir, thank you for the video!
How to select the variant of our gene of interest? On what basis we have to choose?!

[@ogunoluwamayowa4749]

Lovely, thanks

[@alpmogulkoc9148]

thanks really helpful

[@h1bB0ilzZ]

Thanks for the video, it was very helpful. Is the process the same for qPCR primers?

[@IkanPediaMalang]

nice explanation...

[@gehadmahmoud6058]

Very nice explanation. Thank you :)

[@ayeshamalik456]

Hi, thanks for this wonderful Video. I learnt a lot from your videos. Can you please make a video on how to analyze sanger sequencing through Geneious Software and MEGA? Will be much Appreciated. Thanks

[@r.seenaiah]

Good information

[@sanjuktaghosh8802]

Hi, a very useful video. Could you link the video in which you have discussed how to increase stringency so that the primers become more specific. Kindly reply :)

[@somayehmohammadi7794]

Hello .How to be extract PSSM profiles and to be generates matrix? I have consider linear substitution probability matrix.

[@papajiang8487]

I am wondering how does this tool guarantee the primers are specific to human? Can I say once you input the organism option as human, then the tool will help you to design specific primers to human? And if the primers only have one SNP to a different species, does it still call specificity just due to the one SNP?

[@guihuajia7696]

very good video for people!

[@BioResource]

Thank you. Consider subscribing 😊

[@anishamn2172]

How you assign a particular value to Tm. What is the criteria for that value?

[@mohamedsal_traiki6954]

Dankeschön

[@prabhubiograd]

The video must have included probe design too in case of real-time PCR

[@murchanabarua7168]

I have a question? Using this website how would I make a forward and reverse primer that is 18-35 nucleitodes long? because whenever I put that into "PCR product size" from min 18 to max 35 it doesn't work.

[@AZMERANDA24]

Thank you for the video, my question is, what I should do if the primers were not found? Is there another way to design primers?

[@BioResource]

Hi +Dina Human,
You can relax the parameters / stringency or input the region of interest and try using primer3.
Hope this helps.

[@beinghuman4457]

sir i have a confusion ?
when we have to select nucleotide and when we have to select gene in the database ?

[@yossiepdrn8933]

Thanks!(:

[@mohammadrezasamiee5669]

Hi thanks for the video, but I didn't see probe for real time pcr

[@egamberdimaxmudov1676]

How can I record laptop monitor that I design primer ?

[@bijayininayak2100]

How to do primer sequencing in plant gene

[@chinonyeonyeulo7707]

I want to run a primer blast for a microorganisms, how do I run the DNA sequence?

[@raselkhan1384]

Have you figured it out? I'm struggling to validate some of the primers for resistance genes that I collected from literature.

[@yunasali5642]

sir, i want to know about entrez query

[@biologywithboss8296]

Sir some problem please help me RT PCR primer design

[@adeshkolekar3425]

HELLO SIR ITS TAKING TOO MUCH TIME TO GET THE RESULT CAN YOU PLEASE HELP TO SOLVE THIS ISSUE

[@palakmehta8820]

Hello sir.. I am trying to make primer for plant sample. Itried to do as shown in the video but it is showing error like.
The primer specificity can not be determined as sequences for the selected organism are not present in selected database: refseq_mrna please help me with this.

[@Achilles189]

Are you malayali

[@drrakeebahmad8736]

Great

[@BioResource]

Thank you. Consider subscribing.

[@KimiaGhadiri-qu1ev]

Hello , thanks for your explanations .
I wondered if i can ask some questions .
Iam a total newbie in PCR .
I used Perlprimer app for making primer for CFTR gene but the result in NCBI site for my primer was different with the app eventhough i considered the same situation for both can u please help ? 🩵