In our experiment:
Lyophilized Oligo Primers supplied on 50 nmol scale were diluted in 500 µL of 0.1X TE buffer to make it 100 µM stock tube.
[(50 nmol/500 µL TE)×(1000 pmol/nmol)=100 pmol/ µL=100 µM]
Then 1:20 dilution with DEPC treated NFW makes 5 µM working stock. In 20 µl qPCR reaction mix, 0.75 µl of working stock primer quantity gets its final conc. of 0.1875 µM.
[using c1v1=c2v2, c2= (5 µM × 0.75 µl)/20 µl = 0.1875 ≈ 0.2 µM]
- You cannot dilute all of your primers in 500 yl of TE and assume they will all come out at the same concentration
- Different primers are made with different efficiencies so to make 100uM = 100pMoles/ul 'parent stock' you need to customise the amount of solvent you use
- Ordinarily when you have primers synthesised if the company doesn't tell you the exact amount of water/TE to dilute your primers in it should provide the yield of each primer in nMoles
- In that case to make 100uM stock you take that integer and multiply by 10
- Say your primer yield is 25nMoles
- Then to make a 100uM stock you dilute in 25 x10 = 250ul of water or TE
- Hope that helps
Final concentration of 1-2 micromolar in qPCR reaction mix provides good result and while optimized, 0.1875 ≈ 0.2 µM might demonstrate good result.
- You cannot dilute all of your primers in 500 yl of TE and assume they will all come out at the same concentration
- Different primers are made with different efficiencies so to make 100uM = 100pMoles/ul 'parent stock' you need to customise the amount of solvent you use
- Ordinarily when you have primers synthesised if the company doesn't tell you the exact amount of water/TE to dilute your primers in it should provide the yield of each primer in nMoles
- In that case to make 100uM stock you take that integer and multiply by 10
- Say your primer yield is 25nMoles
- Then to make a 100uM stock you dilute in 25 x10 = 250ul of water or TE
- Hope that helps
I agree with Laurence sir answer. Any company in India gives the exact volume TE buffer to be added to primers. That volume never be the same for any primer, not ever for FP or RP.
Thank you Laurence sir for your valuable suggestions which will definitely help me in our work.
regardless of concentration, in a 50 ul PCR reaction you want between 20 and 30 ng of each primer
Dear Amy
whilst your approximation holds since most primers are 18mers to 23mers, it really is about the number of primer molecules, i.e. molar equivalents
In a typical PCR reactions you would start with 1ul of a 10uM primer, i.e. a 1/10 dilution of your 100uM parent stock. this is 10 pmoles of each primer
For a typical qPCR reaction however, as qPCR is much more sensitive than gel based PCR, you would typically use 1ul of 2uM stock in your reaction; that is 2 pmoles of each primer
This is a 1/50 dilution of your parent stock
Note as well: when making up your parent primer to 100uM you add 10mM Tris pH 7-8 or TE
When diluting down primer for actual PCR and qPCR you NEVER dilute with TE as EDTA can inhibit the polymerase. Only ever water
You should keep your 100uM stock at -20C and freeze thaw as necessary
For working primer stocks diluted in water, keep at 4C for upto 2 months, then discard and go back to parent stocks at -20C for future studies
apologies, I assume approx 18mer primers as these are most common. I calculate the molar ratios with the equation pmoles = (X ng * 1000) / (bp length * 650 Daltons)
Is there enough EDTA in a few ul of primers to inhibit a 50 ul reaction? Twist and Genewiz who supply oligos do not recommend re-suspension in water. Adding a touch of Mg also overcomes any tiny quantity of TE inhibition. I have had oligos suspended in MGBW rapidly degrade. I always resuspend in TE, and it doesn't seem to cause problems.
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