Tips on Primer Dilution and Concentration for PCR

[oBWhat]

hello all,

I have some old and long methods to get primer dilution factors for the right concentration for PCR.

Does anyone have some quick and new methods (ie website program) to do this?

thanks!

[richadeo]

when u have lyophilized primer (dried form), add TE buffer (pH 7) to make
concentration 100 µM. Then dilute it to make 10µM working solution. This stock can directly be used for PCR. If u use 1µl of this stock in PCR mix (25µl) u are using 0.4 µM of primer in final mix.
Hope i have solved ur problem.

[coolakul]

Hey I would just like to add, that I found a website with a useful piece of calculation which might help getting a rough estimation as to how much primer you have to use. Although this calculation you do need to know some other variables. Check out this article by Biswajeet -
pharmaxchange.info/press/2011/07/polymerase-chain-reaction-part-i-principle-components-procedure-and-stages-of-pcr/

[sunglassesatnight]

Interesting points. I can offer what works for me, and here it is. Get your lyophilized primers resuspended (in TE buffer or water. I use water and have no problems) to 100uM. To do this easily, take the amount of primer (in nanomoles) and mulitply by 10. then add that many microliters of water (or TE). For example, if you got 34.5nmol of primer, resuspend in 345 microliters. I make my working solutions at 5uM by adding 25uL forward, 25uL reverse, and 450uL of water. Then just add as much primer as is necessary to your PCR mix. If you have no idea how much primer you need, you can run a gradient. Make replicate PCR reactions with increasing amounts of primer.

[Indriyani]

I have not good result of my PCR which primer dimer too strong. I use 10 uM of primer, I have tried also with 5 and 1 um, but still not good.
I have tried for Annealing temperature (55 C) with 30, 20 and 10 sec, but still not good result. Please, your suggestion about this.

[Indriyani]

Dear Sir,

I am doing PCR with target size is 421 bp. Primer concentration is 10 uM, annealing temperature is 55C in 30 sec, 40 cycles. The problem is too strong of primer dimer. Would you please suggest me how to solve this problem?

[butters]

Hi Indriyani, how much did you load for the 10uM primer?

You could try reducing the amount of primer used. Sometime one of the primer (forward or reserve) is causing the problem. So varying primer individually will help, if your lab have RealTime PCR machine. Try look for the manual, there should be one chapter on primer optimization.

good luck.

[Pigous]

In our laboratory we're always dilut our primers. The goal information is the ug quantitity.
For example: my primer had 194,31 ug and I added 194,31 ul of water ( stock). After I performed a dilution for 50 uL (for use) and the concentration final this is 10 pmol wich I always dilute again for 3 pmol. In my PCR reaction with 50 ul as final volume I used always around 3 pmol of primer and I have a good results for cloning directly in expression vector or cloning vector.

best regards

Pigous.

[rizky]

Hi indriyani,

How do you design the primer? I use primer3 software to make the primers.

I dilute the primers 10x with TE buffer. before I runned the primer, it will be better to optimize the primer condition with several annealing temperature.

good luck

[Indriyani]

Thank you for suggestions. Now, I have a good result of my PCR. I designed by using Clustal W and dilute primer with H2O.