How To Create Real-Time PCR Primers Using Primer-BLAST
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- Опубликовано: 13 фев 2018
- This video tutorial is part of the Mastering qPCR Online Course in The Top Tip Bio Academy.
🎓 MASTERING QPCR ONLINE COURSE 🎓
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In this video tutorial, I will show you how to create real-time PCR primers by using NCBI's Primer-BLAST free online software.
The Primer-BLAST software is great since it uses the Primer3 algorithm to design optimal primers, while simultaneously blasting these sequences on the genome. This ensures the primers will not amplify off-target regions.
THE ONLINE GUIDE
toptipbio.com/real-time-pcr-pr...
NCBI WEBSITE
www.ncbi.nlm.nih.gov/
VIDEO BREAKDOWN
Step 1: Finding your gene variant (00:18)
Step 2: Loading Primer-BLAST (01:53)
Step 3: The Primer-BLAST settings (02:22)
Step 4: The primer reports (08:40)
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THE ONLINE GUIDE
toptipbio.com/real-time-pcr-primer-blast/
There is a virus...
Literally the only useful primer blast tutorial on RUclips. Subscribed
Thanks for uploading this video. It's the best, or easy-to-understand qpcr primers video I did watch so far on RUclips.
Thank you very much for your tutorial, very concise and precise
Thank you for making this wonderful lecture.😊😊
This channel deserves more views!!!!
Thank you very much! This helped me a lot!
useful. easy to understand. nice share. Thank you!
Thanks for the feedback
Excelent! Thanks, right to the point and very clear. Cheers from Chile
Glad it helped Fernando!
Thank you very much. This is soooo helpful!!! You saved my life!
You are very welcome
This video is so useful
Thank you so much, this video has released my pressure
Very welcome Rita
What if you use the DNA sequence instead of the full (unspliced) mRNA? Some videos suggested the DNA part, so I am feeling a bit unsure about the mRNA part here that you are suggesting, but I do like the overall video and the online guide--thank you so much.
thank yo so much, it was very useful
Very welcome
great!
Thank you for this video
Very welcome
This is a really useful tutorial!
Q: Why is the maximum PCR product the same as the minimum intron length?
Hello, thank you for this informative video! I need to create miRNA primers, are the features and parameters the same?
awesome
Thank you
You are welcome
thank you unlimited thank to you
hi, if you already have your primers and you input their sequences is there a way to make the primers show as a track on the graphical view without downloading the track first?
Hi, thank you for the excellent tutorial made.
Q: in the case of prokaryotic organisms, since exon/intron do not exist, is it still important to include the intron/exon requirement in the qPCR primer design?
No, in prokaryotic organisms this feature will not work due to the lack of introns. Thanks for your feedback :)
thank you, may i ask you a question? if i want to design a virus gene(plus sense ssRNA, converted to cDNA) primer with ncbi, would species specification to homo sapiens be okay?
nice
Wonderful. I need to design a primer of IL6.. I am lucky...thanks
How to validate the primers that you design using NCBI???
Is there any way to force NCBI Blast to find a primer pair that works for *all* variants of an mRNA?
I was trying to just put the longest variant and filter out the specific ones, but it seems the algorithm tries to be particularly specific and it only finds primers specific for one variant.
It seems "Allow primer to amplify mRNA splice variants" checkbox just before Get Primers button stops this behaviour; but I still can't force it to get pairs that works for all variants.
Edit: Ah, choosing the tab "Primers common for a group of sequences" at the very top of the whole tool seems to be the way to do this.
What if I only get cds sequences when searching for the primer ? I’m trying to design nodC gene for Sinorhizoboum strain, but i didn’t get the full mRNA sequence
very useful thanks Can i design qpcr primers from a complete genomic CDS bcz blast don't have mRNA Seq
Many thanks for the feedback.
Of course, however, you will be unable to design primers that are separated by an intron or span an exon/exon junction when using a CDS as the sequence. So, I would advise you perform a DNase digest step during RNA extraction to reduce the chance of genomic DNA amplification during PCR.
All the best
Steven
How to choose organism in NCBI after nucleotide search..... Most ofter my search for gene and organism never pops up... Kindly help
Can't believe I have done so many qpcr and doesn't even understand exon-exon span until now...
Are you taking mRNA sequence because you're doing reverse transcriptase PCR? How would do the same with genomic DNA, and if your forward primer needs to be upstream of exon 1?
That is correct, this method designs primers based on the mRNA sequence. If you want to do the same for genomic DNA, then you wil need to find the correct gDNA sequence on NCBI first or you can simply copy and paste the FASTA sequence in the Primer-Blast window at the top. You can also specify the range where your forward and reverse primers should bind. So, you can instruct to have the forward primer before exon 1, for example
I'm curious. If taq polymerase can do about 60 nucleotides per second, which would be about 600nucleotides in 10seconds, how would lowering your introns to 200bp keep it from amplifying DNA?
Hi Jennifer. Thanks for the question and good point about the intron size. Obviously the larger the total intron size the better. In some cases, if the intron size is too small, this wouldn't prevent gDNA amplification per say, but it would make it easier to identify gDNA contamination on an agarose gel due to the known size with and without the intron. Just an extra step to help out troubleshooting in the future really. I hope that makes sense and helps. Thanks,
Steven
Why do I need to contain introns in the data? I think we need to design an exon-exon junction primers.
Hi Osama. You can do either spanning an exon-exon junction or separated by a large intron. I had some bad results by using primers that spanned an exon-exon junction. So i usually prefer they are separated by an intron. But that is just my preference. Thanks, Steven
Why didn't you upload the video where you will be discussing the best primer pairs to be chosen
lol, yeah, where is the video .
Sorry this is late! Here you go, I hope it helps:
ruclips.net/video/scX2cFBqs9M/видео.html
What If I have primer variants? How can I choose the best variant? All the genes that I need to get a primer have variants.
Hi Raquel
If there are gene variants then it might be worth reading the literature for studies that have looked at the same gene as you to see if they give an accession number. This will then correspond to the exact variant. Hope that helps. Sorry there is no easy way to know as it is experiment specific
can we use these primers for Qpcr as well?
Of course. These apply to primers using intercalating dyes (SYBR Green etc). There are more considerations when using probe-based variations (eg TaqMan)
Why exon exon spanning option was not selected even though it could help to avoid genomic DNA amplification?
I've had issues with exon-exon spanning primers in the past. But, by all means you can select it to reduce the chance of genomic DNA amplification. Alternatively, have the primers span an intron (and then see if the intron is very large), to prevent gDNA amplification
@@StevenBradburn Thanks for the reply.
I did not understand the part that you explain why not to pick the CDS sequence...this is because you could not put the option in primer-BLAST for exon-exon juntion or span and intron?
That is correct :)
If you use the CDS, then the file does not contain any information about introns. Therefore you will be unable to design primers that span an exon/exon junction, or are separated by an intron
I couldnt reach the 90% ef, even after 2 different primer design
Hi! This works for primer forward and reverse but how to design the probe? Thanks!
Hi Tamara. So this method I show only works for intercalating dye based qPCRs, such as SYBR green. If you want to design primers for probe based assays then it's best to use a different program, such as the online program by GenScript www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool
Just a question: the part about him checking off 'primer pair must be separated by at least one intron on the corresponding genomic DNA' and doing this because we want to avoid genomic amplification, are we checking this off assuming that in our RNA purification, we'll have some genomic DNA contaminant?
Hi Cindy. So i always recommend separating primers by at least 1 intron to limit any DNA amplification. This is just a precaution as you can never be too careful, especially when it's SYBR Green qPCR. You could get DNA contamination during qPCR set up for example.
I hope that helps.
Thanks
Steven
Got it! Thank you so much :)
I followed the same steps as you and did not obtain the window as in 8:47.
the result was a page named "PREDICTED: Homo sapiens interleukin 6 (IL6), transcript variant X2, mRNA" with no primers listed.
@@sayuriyamagata6853 It seems like you may have selected a different target in Primer-BLAST. This vieo designs primers for the Homo sapiens interleukin 6(IL6), transcript variant 1, mRNA. Is the accession number at the top NM_000600.4?
I'm pretty new at gene cloning and am doing a biotech experiment for a science fair at my school. Since you seem to know what you're talking about, I have a few questions for you that I hope you can answer.
To provide you with some context, I am going to remove a string of genes from a bacteria that code for proteins that aid in cellular mineralization. Said bacteria is Bacillus subtilis 168, and its entire circular chromosome has already been sequenced. The adjacent genes I will be removing are: etfA, etfB, fadB, and fadR. You can view them here: subtiwiki.uni-goettingen.de/v3/gene/view/251600E5FD52DC0352A123F1224C037577F5A09C. You can click the button that says "sequence" on each gene and the database will show you the nucleotide sequence. I'll be using the restriction enzyme BspDI to cut the DNA at restriction sites that flank my genes of interest (the sites are located in the abf2 and lcfA genes and the cutting sequence is ATCGAT ).
My first question is: what is the difference between Real-Time PCR and conventional PCR? I believe that I will be conducting conventional PCR since I will be using gel electrophoresis, ethidium bromide, and UV light to visualize my fragments. Moreover, the fragment that I will be working with is well over 2kb long, and you mentioned in your video that RT-PCR fragments are optimally 70 bp - 200 bp long.
2nd question: I want to include restriction sites (for BspDI) on the 5' end of my primers, and since I know the exact locations of these sites as they appear in the bacteria's DNA (as I mentioned, the sites flank my genes of interest), I know exactly where I need my primers to anneal to. Is it possible for me to just design my primers on my own so long as they are an adequate length and fulfill all GC concentration and Tm requirements? I feel like this program generates primers in nonspecific locations while I need primers that bind to specific regions on either end of my genes of interest. FYI, I need to use PCR to amplify these genes b/c I will be cloning the insets into a vector for transformation.
3rd question: Since bacteria are prokaryotes, they have no introns in their DNA, only exons. Knowing this, can I just ignore all of the sections about exon junction spans, exon junctions match, or intron inclusion?
4th question: If I end up using this NCBI software to design my primers, my best bet is that I will use the "Range" feature to position my primers at exact locations around my genes of interest and the "Primer Parameters" function to insert my own forward and reverse sequence. This being said, after completing the design does one order the primers from NCBI or from somewhere else? Also, do you know the approximate cost of the primers?
I know this is a lot but I would really appreciate your help. Thank you in advance.
If you want a response, it's better to go to Research gate. If you input "research gate" in your URL browser. It is a .net website. Many faculty members in my college use this, and I believe it's world wide, so different professionals can answer your question. It's trustworthy, and super helpful.
@@SanguinaryStrife ok, I'll give it a try
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For mRNA, even the longest transcript mRNA does not contain introns, it's all exons. I think there is a mistake in your expression concerning the choosing of transcript.
Hi Lanxin,
The mRNA sequence in NCBI contains information regarding introns, unless you select the CDS version (which only contains exons).
Thanks
Steven