How to design Primers and make primer mixes - KravitzLab/KreedLabWiki GitHub Wiki
What are PCR primers ?
Primers are small synthetic DNA sequences that will serve to start and end the DNA sequence of interest
They come in forward (Primer 1) and reverse (Primer 2). To make sure that you are looking at the correct gene, it's crucial to have a specific set of primers for your DNA sequence of interest, this make the designing of primers a "Non fatal critical step"
Why critical ?
You need to have specific primers sequences or else you will be amplifying "noise" or off-target genes in your PCR and your results will be non exploitable.
Why non fatal ?
Designing primers is surprisingly easy, takes a few minutes, doesn't cost a lot of money and having the wrong set of primers for your qPCR just means that you need to re-order new ones but it won't mess up your experiments, you'll just lose a few days for the time of delivery
So if you do your qPCR with the wrong set of primers and only realize it 2 weeks later, if you still have some cDNA, you'll be fine, you will just be frustrated to have lost that much of time.
How do I choose good sets of primers ?
There are 3 ways to have new sets of primers. The "collaboration" way the "lazy" way and the "I design my own primers" way.
- The collaboration way : Just find a researcher in your lab/team that has ever used to do qPCR for your genes of interest and ask to borrow some of their primers. Since primers are extremely cheap and very easy to make a stock of, they will very likely say yes.
Caveat : If you are the only one doing qPCR then it's a bit of a problem.
- The lazy way : Find a (preferentially recent) paper where they used in the same strain of mice and with similar qPCR parameters a set of primers of your gene of interest and use their primers sequence.
Caveat : Still check the specificy of the primer sequences (See below)
- I design my own primers way : Use primerblast to make your own primers.
Caveat : It may sounds a bit intimidating or tricky but tbh it's pretty easy.
How do I design my own primers ?
Ok let's say you want to design primers for the Tyrosine hydroxylase gene (gene that makes the tyrosine hydroxylase, enzyme that synthesize dopamine) in mice.
1- Go to NCBI : https://www.ncbi.nlm.nih.gov/
2- On NCBI go on "Gene" and search for "Tyrosine hydroxylase mus musculus"
3- Click on the tyrosine hydroxylase, you'll get on the page of the gene
4- Take the Refseq Select (NM_009377.2)
5- Go on primerblast : https://www.ncbi.nlm.nih.gov/tools/primer-blast/
6- In the "PCR template" area, put the Refseq select number
7- In PCR product size, put "70 to 200 bp", that's the ideal size for qPCR
8- Set primer melting temperature at min 59, opt 62, max 65 (This depends on your PCR set up, for 60°c of annealing, that's the ideal set up, see the page about setting up the qPCR parameters)
9- In Exon junction span select "Primer must span on Exon-Exon junction", its to increase the specificity of the primers
9- Organisms choose "Mus musculus"
10- Click on "Get primers". This can take a few minutes
11- Then you get a list of "Primer pairs"
12- Optimal primers have a GC content of 40-60%, so choose the adequate primers. It's possible to have several primers sequences that fit the criteria, just choose one.
13- Order the primer sequences (Forward and Reverse) and Voilà !
14 - ADDITIONAL STEP BEFORE ORDERING : It never hurts to verify the primer sequence. Go back to primer blast, put the "Forward" and "Reverse" sequence that you chose, choose "mus musculus" for organism and click on "Get primers"
15- NORMALLY, primerblast will be able to identify this specific primer to the DNA sequence, then you will be able to be sure that your primers are specific !
How to make the primers mix ?
1- The primers your receive (IDT brand) are in powder, they tell you how much RNAse free water you need to add to the tube : Look for the "For 100 uM, add X uL" line in the sheets that arrive with the primers. That will make your primers STOCK for the FORWARD AND the REVERSE primer (You get one tube for forward, one tube for reverse)
2- For the primers mix, put 490 uL of RNASe free water in an eppendorf.
3- Take 5uL of the FORWARD stock, put it in the eppendorf
4- TAKE A NEW TIPS, I CANNOT STRESS THIS ENOUGH !!! And take 5uL of the reward and put it in the eppendorf
5- Gently mix the eppendorf
6- You are done !
7- Keep the primers (stock and eppendorf) in the fridge at -20°c for optimal conservation.
Common issues with the PCR primers and their solutions
Oh my god, I forgot the primers solution (Stock and eppendorf) on the bench the entire weekend !
Yeah, they're fine. They are DNA sequences. They will be fine for months at room temperature. We keep them at -20°c to avoid bacterial proliferation and paranoia. A few days on the bench are not a big deal.
I have very weird results on my qPCR. When looking at the melting curve, I see a peak at low temperature.
It's probably primer-dimer contamination.
If you didn't change your PCR parameters and the primers worked before, it's likely pipetting mistake or cross contamination
- Pipetting mistake : You put too much primers. Just start a new eppendorf and re-run the PCR
- Cross contamination : You put the forward sequence primers in the reverse tube by not changing tips or whatnot. It's not a big deal, but you'll need to order new primers.
I have no amplification but I know my primers are working because they were working yesterday !!
Likely it arise from 2 problems
- You forgot to add the primers in the PCR tube. When you pipette so much, it's very easy to forgot to add the primers.
- You took the primers from the eppendorf before it was completely thawed. Therefore you only took the thawed liquid that contained much less primers mix, just redo the PCR with the thawed primers eppendorf and gently vortex it for good measure.
Can I use old primers solutions that have been in the fridge for 2 years ?
- Likely yes. Run a qPCR "test" with them on not so important sample but it's likely fine. Again, these are DNA sequences.