In our lab we dilute all primer stocks to an initial 100uM concentration. These are our stock tubes. We then make 1:10 dilutions for our working stocks meaning that each primer is at 10uM. If your primer comes with a QC sheet, it should tell you how many total nmol are in the stock tube. Our primers generally come lyophylized. Therefore, to make our 100uM stock, we just move the decimal over one place on the total nmol concentration and add that much 0.1xTE. For example, if your stock comes will 33.1nmol of lyophylized primer, we would add 331ul of 0.1xTE. This is our stock tube. We then make a 1:10 dilution from this using ddH2O for our working stock. For a 25ul reaction, we would generally start with 1ul of each primer working stock. However, if you truly want to find your optimal concentrations, then you would need to do a primer optimization titration. For this we usually try concentrations from 50nM all the way up to 900nM for each primer in all possible combinations. It is a pain, but it is amazing how often your optimal concentrations for each primer will be very different from each other. Hope this helps

In our lab we usually have the primers at 200μM master stocks. I keep my working aliquots at 10 μM. Τhe most usual final forward/ reverse primer concentration for qPCR is 250nM (for end-point PCR it could be higher). Therefore, for a 20μl reaction, I'd put 10μl 2x master mix, 0.5μl forward primer, 0.5μ lreverse primer and the difference to 20 - cDNA and water.

It's very simple to calculate. Before that I would like to ask u one concentration.

Wts d stock concentration of primer?

Wts d total volume of ur PCR reaction?

If you know these two than u can easily calculate ur final concentration of ur pcr reaction. Basically 1 to 2 micro molar is enough for a single reaction

X= final con if the primer x total volume of the reaction

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Stock concentration of the primer

All d best!!

Hi,

As fer as i am concern, I generally use 1 ul (10 micro molar conc) of each primer (forward and reverse) at the time of standardization. most of the time it works out well. Depending on your experiment you may need to change the volume of the primer. Now what about he concentration, as I already said I always prefer 10 micro molar concentration of working primer solution.

you have re suspend your stock primer to 100 micro molar concentration then use the formula of n1v1 = n2v2. When you are re suspending your primer, just check the label, if it is saying that it is 42.5 nano mol then add 425 ul rnase free water. vortex it and short spin it. now you got 100 micro molar stock primer . then make 10 micro molar working primer and use 1 ul of each primer for your PCR.

Best of luck. I hope that it would be useful.

I completely agree with Bhaumik, in 25 microliter PCR reaction mix, 1 microlite of 10 micro molar concentration is good for most of the reactions. In our Lab also each and everyone is using same concentration and are getting good results.

In our lab we dilute all primer stocks to an initial 100uM concentration. These are our stock tubes. We then make 1:10 dilutions for our working stocks meaning that each primer is at 10uM. If your primer comes with a QC sheet, it should tell you how many total nmol are in the stock tube. Our primers generally come lyophylized. Therefore, to make our 100uM stock, we just move the decimal over one place on the total nmol concentration and add that much 0.1xTE. For example, if your stock comes will 33.1nmol of lyophylized primer, we would add 331ul of 0.1xTE. This is our stock tube. We then make a 1:10 dilution from this using ddH2O for our working stock. For a 25ul reaction, we would generally start with 1ul of each primer working stock. However, if you truly want to find your optimal concentrations, then you would need to do a primer optimization titration. For this we usually try concentrations from 50nM all the way up to 900nM for each primer in all possible combinations. It is a pain, but it is amazing how often your optimal concentrations for each primer will be very different from each other. Hope this helps

Commonly the standard PCR reaction required 5 to 10 pmole concentration of each primer. So to bring this one you have to refer the data sheet provided by the primer manufacturer. in data sheet they given the how much micro litter you have to add to bring 100 micro mole concentration. if you added now the final concentration per micro litter is 100 pico mole. for 10 pico mole concentration of primer for standard PCR from that stock you have to take 1 microlitter of each primer for 100 micro litter reaction.

go through this link for full explanation

http://bitesizebio.com/9945/how-to-calculate-a-dna-primer-concentration/

From lyophilized powder

Primers purchased from companies will often be sent as a lyophilized powder, and the company will tell you the nmoles of primer they have provided.  If you are confident that you have recovered all of the powder and have had no previous issues with the amount of primer sent, then a quick resuspension and calculation can be done without measuring the OD of the primer.  If your downstream application with the primer is highly sensitive to primer concentration, or you are unsure of the quality of primer, you should measure the OD of the primer after resuspension and use the second method for calculation.

1)   Resuspend the primer in 100 ul of water or buffer and use the following calculation:

(XX nmol/100 ul) x (1000 pmol/nmol)= pmol/ul = uM

Example: (22 nmol/100 ul) x (1000 pmol/nmol) = 220 pmol/ul = 220 uM

2)   Alternatively, you can quickly resuspend a primer at 100 uM by resuspending the powder in a volume in microliters that is 10x the number of nmol.

Example: Resuspend the 22nmol of primer in 220 ul=100 uM

].

In our lab we resuspend the primer to a concentration of 100 µM by resuspending the powder in a volume in microliters that is 10x the number of nmol.

Example: Resuspend the 20nmol of primer in 200 µl=100 µM