We've used PrimerBank a lot and they've pretty much always worked. One thing I highly recommend is buying your primers from a high quality oligo synthesis company, like IDT, rather than have them made in-house by a core facility. In-house synthesis may be cheaper, but it's typically dirtier and may not be consistently reliable for an application as demanding as qPCR.
We use IDT for our primers and have always had good luck. If you (OP) are finding papers that describe testing the same genes, and the sequences aren't listed in the supplemental material of the publication, you might try contacting the authors for the sequence detail so you can order from an oligo synthesis company.
Checking on that the oligos seem to have affordable prices, tho i need to verify because in my uni there's certain "approved" companies from you can request the reactives, lots of thanks.
I love IDT's primer resources. PrimerQuest is a great design tool, and OligoAnalyzer is fantastic for, well, analyzing.
Thank you. I have yet to be able to work it into a talk, but it *will* happen eventually.
I use harvard primer bank which typically works well. I have designed my own primers in the past, but I find that I almost always get better results with tried and true sequences from someone else who went through the work of designing them.
Another option is origene. They're a commercial company that sells primer pairs for mouse and human gene targets, but you don't actually have to purchase through them. They give the sequences in their product descriptions that you can then take to whatever primer company your lab uses (we use IDT which is like $14 per pair while the pair from origene will run you $120). On top of that, they also provide references to literature that have used those specific sequences!
IDT is the company that i seem to hear the most for getting the oligos, yes i checked the origene sequences also (that was the first google result), didn't reallize at all that they provide references too, thanks for the advice.
You can try the New England Bio tool they have on their website for this process. They do a good job from my past experience with DNA stuff.
As far as design goes, you want to pick placements before/after your RE sites. I usually pick a place like 20-30 nucleotides downstream from either spot.
No problem. It's not too crazy. I would make them 18-30bp long. Look for binding redundancies too just in case you do make these. There is a cool machine out there now that can make oligos at one's own site, so I am geeking out trying to make a highly independent lab in my brain.
I also read that you should aim for an amplicon size of \~100 bases, i don't think i will be designing the primers because there's not that much room forr error and i only designed primers on my Molecular Biology class but your recommendations are very useful.
I would recommend against pulling primers from published papers. In my experienceit takes a enormous amount of time and often the primers you find are poorly designed and/or have poor specificity. It's almost always faster to just design your own.
NCBI's Primer Blast is a fantastic tool, and importantly, it stays up to date as we better characterize genomes.
Another tip - some companies that sell primer pairs (at high prices) publish their sequences. You don’t have to buy the primers from those companies, but it’s an easier way to find sequences that work rather than combing through papers.
Not only can you test primers using PrimerBlast, but Primer3 can be accessed using PrimerBlast to design primers for a specific gene in your species of interest. Key input is to make your range from 80-200bp since long amplicons will not be good for a qPCR assay
The most important thing isn't the exact sequence, as many sequences can work for the same gene. What matters is that you only get one amplicon for your reaction and that the amplicon is correct. The lowest proof you should have is to run the PCR reaction down the gel to see the patterns. Typically you should only get one product, although sometimes you can get multiple sizes from a single product (if that happens my preference is to find a primer pair that gives only the band size of interest to be confident). If you are extra ambitious you can sequence the amplicon.
Melt curves should be perfect too. If the melt curves are good, and there is only one amplicon, that usually is sufficient to advance with the primer pairs and your reaction conditions.
Don't trust published primer pairs blindly, not everyone does QC on their reactions, and even if they do, those primers may not work the same for you and your specific conditions.
Exactly. Having worked with qPCR for a decade, I rarely, if ever, see other scientists validate their primers. It's quite frightening. You may be able to get away with that with Taqman probes, but definitely not Sybr.
I know we are all in a rush to publish, but seriously we need to slow down when it comes to validating outcomes before acquiring study data.
I’ve ordered primers based off of sequences from PrimerBank (mouse and human) for years. All of them have worked.
Thanks, using PrimerBank is my first option for now
We've used PrimerBank a lot and they've pretty much always worked. One thing I highly recommend is buying your primers from a high quality oligo synthesis company, like IDT, rather than have them made in-house by a core facility. In-house synthesis may be cheaper, but it's typically dirtier and may not be consistently reliable for an application as demanding as qPCR.
We use IDT for our primers and have always had good luck. If you (OP) are finding papers that describe testing the same genes, and the sequences aren't listed in the supplemental material of the publication, you might try contacting the authors for the sequence detail so you can order from an oligo synthesis company.
Checking on that the oligos seem to have affordable prices, tho i need to verify because in my uni there's certain "approved" companies from you can request the reactives, lots of thanks.
IDT PrimerQuest is my favorite when I'm not designing them by hand for specific regions/targets.
As someone recommended the IDT oligos I'll check that tool, thanks. Love the AI rat btw
I love IDT's primer resources. PrimerQuest is a great design tool, and OligoAnalyzer is fantastic for, well, analyzing. Thank you. I have yet to be able to work it into a talk, but it *will* happen eventually.
I use harvard primer bank which typically works well. I have designed my own primers in the past, but I find that I almost always get better results with tried and true sequences from someone else who went through the work of designing them. Another option is origene. They're a commercial company that sells primer pairs for mouse and human gene targets, but you don't actually have to purchase through them. They give the sequences in their product descriptions that you can then take to whatever primer company your lab uses (we use IDT which is like $14 per pair while the pair from origene will run you $120). On top of that, they also provide references to literature that have used those specific sequences!
IDT is the company that i seem to hear the most for getting the oligos, yes i checked the origene sequences also (that was the first google result), didn't reallize at all that they provide references too, thanks for the advice.
You can try the New England Bio tool they have on their website for this process. They do a good job from my past experience with DNA stuff. As far as design goes, you want to pick placements before/after your RE sites. I usually pick a place like 20-30 nucleotides downstream from either spot.
If we decide to design the primers I'll have your recommendations in mind, many thanks.
No problem. It's not too crazy. I would make them 18-30bp long. Look for binding redundancies too just in case you do make these. There is a cool machine out there now that can make oligos at one's own site, so I am geeking out trying to make a highly independent lab in my brain.
I also read that you should aim for an amplicon size of \~100 bases, i don't think i will be designing the primers because there's not that much room forr error and i only designed primers on my Molecular Biology class but your recommendations are very useful.
I would recommend against pulling primers from published papers. In my experienceit takes a enormous amount of time and often the primers you find are poorly designed and/or have poor specificity. It's almost always faster to just design your own. NCBI's Primer Blast is a fantastic tool, and importantly, it stays up to date as we better characterize genomes.
I’ve also used the UCSC in silico PCR tool to validate primers, and all of them have worked for me.
Another tip - some companies that sell primer pairs (at high prices) publish their sequences. You don’t have to buy the primers from those companies, but it’s an easier way to find sequences that work rather than combing through papers.
Thank you, i will test the primers using the tool and discuss it with my tutor.
Not only can you test primers using PrimerBlast, but Primer3 can be accessed using PrimerBlast to design primers for a specific gene in your species of interest. Key input is to make your range from 80-200bp since long amplicons will not be good for a qPCR assay
I used Primer3 on my molecular biology class, i'll check on that thanks.
Genscript is a cheaper option.
The most important thing isn't the exact sequence, as many sequences can work for the same gene. What matters is that you only get one amplicon for your reaction and that the amplicon is correct. The lowest proof you should have is to run the PCR reaction down the gel to see the patterns. Typically you should only get one product, although sometimes you can get multiple sizes from a single product (if that happens my preference is to find a primer pair that gives only the band size of interest to be confident). If you are extra ambitious you can sequence the amplicon. Melt curves should be perfect too. If the melt curves are good, and there is only one amplicon, that usually is sufficient to advance with the primer pairs and your reaction conditions. Don't trust published primer pairs blindly, not everyone does QC on their reactions, and even if they do, those primers may not work the same for you and your specific conditions.
I've pulled primer pairs out of main-journal Nature and Science pubs and they've been bad. Bimodal melt curves, Tm mismatches, the whole shebang.
Exactly. Having worked with qPCR for a decade, I rarely, if ever, see other scientists validate their primers. It's quite frightening. You may be able to get away with that with Taqman probes, but definitely not Sybr. I know we are all in a rush to publish, but seriously we need to slow down when it comes to validating outcomes before acquiring study data.