hello,
I was reading the following paragraph in materials and methods:
"To evaluate the trans-cleavage activity of Cas12f1 RNP with different guide RNAs, a reaction buffer was prepared by mixing 12 µL of 5 µM Cas12f1, 3 µL of 10 µM tracrRNA (either truncated or untruncated) and crRNA, or sgRNA, 6 µL of 100 µM Texas-12 T-BHQ2 reporter in 600 µL of 1x NEB r2.1 buffer. Cas12f1 was activated by adding 5 µL of 1 µM target/water to 95 µL of the prepared reaction buffer. Fluorescence was measured using an ID3 plate reader with excitation/emission wavelengths of 570 nm/615 nm at 46 °C for 1 h."
i am getting cnfused for the conentration of different compounds: is it 5 uM cas12f1 means 5 uM/L or 5 uM/ul?
thank you for the help.
5 uM specifies concentration and it does not matter whether you have a microliter or a liter of it - the concetration (the number of molecules) per unit volume is always the same.
5 uM solution has 5 umoles of the molecule in one liter (that is how it is defined). So, in half a liter you have 2.5 umoles, in 200 ml you have 1 umole but it is still a 5 uM solution. Is that clear?
thank you for your answer, i got confused because I wanted to know the final concentration for each component. if I have protein with concentration of 120 ng/ul. how much will be the molarity or concentration in moles?.
You need to know the molecular weight of your protein - that is how much one mole of your protein weighs in grams (lets call it X). If you know the amino acid sequence of your protein, just paste it into an online program that will calculate X for you. Then, you will know that to have 1 molar solution you need to have that amount of that protein (X=1 mole) in one liter. As you have 120 ng/ul, you would have 120 mg/L. So, when X g in one liter is 1 M, you have to calculate what is the molarity of the protein solution that has 120 mg/L: molarity of your protein = 0.0012/X.
Thank you so much for your help. i found this tool:
https://www.bioline.com/media/calculator/01_04.html
and it can calculate between molarity and concentration