Hello Rafika Nanda Putri

I have gone through the protocol of Sensifast Probe No-ROX One-step kit. You can use the probe of 5uM concentration. Let me explain.

In the preparation of real-time RT-PCR Master mix the final volume of the Master mix is 20 ul.

In the protocol the probe concentration used was 10uM (stock). But You have a stock of 5uM probe.

In the protocol 0.2ul of 10uM probe stock was used to make a final Master mix of 20ul where the final concentration of the probe in the Master mix is 100nM.

Since you have 5uM of the probe stock, you will have to use 0.4ul of the probe from your stock of 5uM to be added along with other ingredients in the Master mix, and make up the final volume of Master mix to 20ul by decreasing the volume of water only and not changing the volume of other ingredients. So as per the protocol you will have to add 3.4ul of water instead of 3.6ul of water.

Let me explain how I arrived at 0.4ul of the probe.

Use the formula N1V1=N2V2

Where

N1= the stock concentration which you have. (5uM) = 5000nM (conversion of units from uM to nM)

V1= the volume to be added from the 5uM stock in the Master mix. (Say X).

N2= the final concentration of the probe in the master mix which is 100nM (as per the protocol).

V2= the final volume of the master mix which is 20ul.

Therefore

5000 x X = 100 x 20

X = 100 x 20/5000 = 0.4ul

Therefore, you will have to add 0.4ul of the 5uM stock probe which you have to the Master mix of 20ul final volume.

I hope this will be helpful.

Good Luck.

Thank you so much for your explanation Mr. Malcolm Nobre , This is very helpful, because previously I also think to take as much as 0.4 uL of stock 5 uM, but I am still not sure how much water should be added. I'll try it. Thank you.

-Rafika

Hello Rafika

Wish you Good Luck for your experiments.

Regards

Malcolm