# 2 fold dilution calculator

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Real-time PCR, also called quantitative PCR or qPCR, can provide a simple and elegant method for determining the amount of a target sequence or gene that is present in a sample. Its very simplicity can sometimes lead to problems by overlooking some of the critical factors that make it work. This review will highlight these factors that must be considered when setting up and evaluating a real-time PCR reaction.

C t threshold cycle is the intersection between an amplification curve and a threshold line Figure 1B. It is a relative measure of the concentration of target in the PCR reaction. Many factors impact the absolute value of C t besides the concentration of the target.

## Dilution Factor Calculator

We will discuss the most common template-independent factors that can influence C t and describe how to evaluate the performance of a real-time PCR reaction. Figure 1, above, shows several parameters of the real-time reaction amplification plot. The exponential phase in Figure 1B corresponds to the linear phase in Figure 1C. The threshold must be set in the linear phase of the amplification plot in Figure 1C.

The C t value increases with a decreasing amount of template. However, artifacts from the reaction mix or instrument that change the fluorescence measurements associated with the C t calculation will result in template-independent changes to the C t value. Therefore, the C t values from PCR reactions run under different conditions or with different reagents cannot be compared directly. The fluorescence emission of any molecule is dependent on environmental factors such as the pH of a solution and salt concentration.

Note that the fluorescence intensity is higher in Master Mix A even though the target, probe, and ROX dye concentrations are the same in both cases. Figure 2. Raw fluorescence data obtained from one assay using two master mixes with the same ROX level.

The difference in signal is due to the master mix composition. The x-axis shows the emission wavelength of the fluorophore and the y-axis shows the intensity of the emission. Note that the baseline fluorescence signals, in a template-independent factor, are different for the two master mixes Figure 3A. Variations in the C t value do not reflect the overall performance of the reaction system Figure 3B. Master mixes with equivalent sensitivity may have different absolute C t values.

Figure 3. A Rn is plotted against cycle number and the baselines for both reactions are shown. The threshold green line is set at the same level for both master mixes. The new C t value obtained by lowering the level of ROX dye has no bearing on the true sensitivity of the reaction, but can have other unintended consequences.

Low concentrations of ROX dye can result in increased standard deviation of the C t value, as shown in Figure 4. The greater the standard deviation, the lower the confidence in distinguishing between small differences in target concentration see the precision section below. Figure 4. Decreasing the ROX dye concentration gives an earlier C tbut increases the standard deviation. The efficiency of a PCR reaction can also affect C t. A dilution series amplified under low efficiency conditions could yield a standard curve with a different slope from one amplified under high efficiency conditions. In Figure 5, two samples X and Y amplified under low and high efficiency conditions show different C t values for the same target concentration. In this example, although the high-efficiency condition the blue curve in Figure 5 gives a later C t at high concentrations, it results in better sensitivity at low target concentrations.

The PCR efficiency is dependent on the assay, the master mix performance, and sample quality. However, this is not true if different instruments, reagents, primers and probes, or reaction volumes are involved in producing the two C t s. Therefore, the absolute C t value comparison is only meaningful when comparing experiments using the same reaction conditions as defined above. Figure 5. Variation of C t with PCR efficiency. Amplification of quantity Y gives an earlier C t under low efficiency conditions green compared to the high efficiency condition blue.Physiology Web.

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Either enable JavaScript in your browser or use another computer in which JavaScript is enabled. Dilution Factor Calculator - Cells per Volume. Meant to be used in both the teaching and research laboratory, this calculator see below can be utilized to perform dilution factor calculations when working with solutions having cells per volume i. These calculations are commonly performed when working with culture media containing living cells such as bacterial cells or mammalian cells.

See also our Cells per Volume Dilution Calculator. When a concentrated solution is diluted, the dilution factor may be expressed as the ratio of the concentration of stock solution to the concentration of the diluted solution. The dilution factor may also be expressed as the ratio of the volume of the final diluted solution to the initial volume removed from the stock solution.

See below for the dilution factor equations. For this particular dilution, it can also be said that the stock solution was diluted fold. It can also be said that the stock solution was diluted fold. Therefore, a fold dilution is the same as a dilution factor of Thus, dilution by any factor X is equivalent to X -fold dilution. If you wish to convert these units to other volume units, please use our Unit Conversion Calculator. Additional dilution factor calculators are also available and are suited to more specialized applications see here.

Each calculator cell shown below corresponds to a term in the formula presented above. Two main options are available for this calculator: the dilution factor may either be calculated or specified. Dilution factor calculated : For this calculation, enter appropriate values in all cells except the dilution factor and the one you wish to calculate. Therefore, at least three cells must have values, and the dilution factor and the parameter you wish to calculate must be blank.

The value of the dilution factor and the blank cell will be calculated based on the other values entered. After a calculation is performed, the calculated cell will be highlighted and subsequent calculations will calculate the value of the highlighted cell with no requirement to have a blank cell.

However, a blank cell has priority over a highlighted cell. Dilution factor specified : For this calculation, the following must be entered: 1 desired dilution factor; 2 either the stock concentration C 1 or final concentration C 2but not both; and 3 either the volume from stock solution V 1 or final solution volume V 2but not both. Therefore, two cells must be blank: C 1 or C 2 and V 1 or V 2. The value of the blank cells will be calculated based on the other values entered.

After a calculation is performed, the two calculated cells will be highlighted and subsequent calculations will calculate the values for the highlighted cells with no requirement to have two blank cells. However, two blank cells have priority over two highlighted cells. For convenience, this calculator allows you to select different volume and concentration units, and the necessary conversions are carried out for you to obtain the value of the blank cell s in the desired unit.

Dilution factor calculated. Dilution factor specified. Dilution factor is calculated. Enter values for any three of the four parameters shown below. The blank parameter and dilution factor will be calculated.

Physiology Web at www. Test Questions. Daily Quiz. Physiology Tutor.Physiology Web. Note: Some functions of this site require your browser to support JavaScript. JavaScript is not enabled in your browser. Without JavaScript, you will not be able to use some features of this site. Either enable JavaScript in your browser or use another computer in which JavaScript is enabled.

Dilution Factor Calculator - No Unit. Meant to be used in both the teaching and research laboratory, this calculator see below can be utilized to perform dilution factor calculations.

When a concentrated solution is diluted, the dilution factor may be expressed as the ratio of the concentration of stock solution to the concentration of the diluted solution. The dilution factor may also be expressed as the ratio of the volume of the final diluted solution to the initial volume removed from the stock solution. See below for the dilution factor equation. For this particular dilution, it may also be said that the stock solution was diluted fold.

### Molecular Biology Dilution & Solution Calculators

As another example, a 2-fold dilution is the same as a dilution factor of 2. Therefore, dilution by any factor X is equivalent to X-fold dilution. If you wish to convert these units to other volume units, please use our Unit Conversion Calculator.

Additional dilution factor calculators are also available and are suited to more specialized applications see here. Each calculator cell shown below corresponds to a term in the formula presented above.

Enter appropriate values in all cells except the one you wish to calculate. Therefore, at least two cells must have values, and no more than one cell may be blank. The value of the blank cell will be calculated based on the other values entered.

After a calculation is performed, the calculated cell will be highlighted and subsequent calculations will calculate the value of the highlighted cell with no requirement to have a blank cell.You probably wouldn't want to dilute by more than a factor of in a single step, because that would give you a large volume of dilute solution.

We can add 0. Call this "Solution 1". How do we dilute a bacterial culture fold, fold, and fold? Chemistry Solutions Dilution Calculations. Ernest Z. Jul 15, You could do the dilutions in one step, or you might have to do serial dilutions. Explanation: There is a limit to how much you can dilute a sample in one step.

## Serial Dilution Calculator and Planner

Rather, you would use the serial dilution technique. This time we add 0. Call this "Solution 2". Related questions How do you calculate concentration from absorbance? How do you calculate concentration from titration? How do you calculate dilution factor? How do you calculate serial dilutions?

How to calculate concentration of solution when it's diluted? What are some examples of dilution calculations? What would be the concentration of a solution made by adding mL of water to What would be the concentration of a solution made by diluting What is the molarity of a solution that is made by diluting What volume of water would you add to See all questions in Dilution Calculations.

Impact of this question views around the world. You can reuse this answer Creative Commons License.The resulting solution contains […] Dilution refers to make a lower concentration solution from higher concentrations. The process is exactly the same as the 32oz way. Serial Dilution Calculator: Amazon. This experiment will then require 6 test tubes, one for each of the dilutions. You must do this to find the dilution factor which yielded your CFU count.

After incubation, colonies are counted on the plate. The dilution factor is the inverse. This problem may be illustrated as follows: Figure 4. Grab a pipette of an appropriate size. The starting concentration is 10 M and the dilution factor is 4.

Related Articles. Calculate the total dilution in a serial dilution. Prime Einkaufswagen. Here, we provide you with every imaginable piece of information regarding serial dilutions; from calculations for the required volume of solution at the end of each dilution, to the exact amount of stock solution and dilutant needed to make the first solution, to the dilution factor of the first nine solutions with respect to the starting solution.

Dilution factor is the total amount of solution per aliquot volume. How to Calculate RF Value. Therefore, dilution by any factor X is equivalent to X-fold dilution.

For our experiment we are using 9 cm3 of each dilution, with 3 repeats. Multiply the individual dilution of the tube X previous total dilution. It is normally a good idea to draw out dilution problems until you are comfortable doing them. Dilution Factor Calculator. The dilution or dilution factor is the initial volume divided by the final volume.

Then we take oz and divide that by 6 and we get When a solution's concentration is reduced, it is called dilution. The 10 represents the total size of the final sample. This calculator is intended for laboratory scale applications although it can also be used for The factors then can easily be multiplied to give an overall dilution factor.

Find dilution factor with initial and final volumes using this calculator. Remember to use clean pipettes for both and to stir the solution thoroughly but gently. It is the ratio of the final volume to the initial volume.My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines.

During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTapand share my knowledge on the topic here to help as many people as possible.

You have 1 part of sample the blue boxes above for 4 parts of water the red boxes above. The way you calculate the dilution factor is the following: you need a number that converts the proportion of sample you initially have to the total volume you will have in the end. By what number to I need to multiply to get to the final volume 50mL starting from the initial volume 10mL? To get from 1 part to 5 parts you need to multiply by 5.

Similarly, in S:T format you already have your sample to total proportionwhich already tells you have to multiply by 5. You can also do the calculations in our dilution factor calculator. A slightly more difficult example. What happens if instead of having 1 part of sample you have more? By what number do I have to multiply to get from 2 to 5? The trick is, you have to divide by the number of parts of sample! For S:T, divide T by S. This example explains the basic steps to use dilution factors forward and backward. Using them forward means finding the cell density after all the dilutions are performed, starting from the original solution.

Using them backwards means finding out the original cell density, starting from the most diluted one. We want to find the cell density of 3 without redoing the calculations with cells, and using the previously calculated cell density. We can now apply it to the original cell density: 0. Same thing for the dilution from 3 to 5 : the cell density of 3 is 0.

We divide the cell density by the dilution factor and we get: 0. Now we can do them backward: The cell density of 5 is 0. In order to get the one of 3we have to multiply by the dilution factor: 0. To get the density of 1 form the one of 3multiply again by the dilution factor: 0. If you want to skip one of the steps i. In the previous example there was no loss of material we started with 11 cells and we made it to the end with the same 11 cells. But what if we split the sample at some point? Then the number of cells in our sample is going to change.

To calculate the number of cells you have in each, multiply the concentration by the volume: 0. Now, back to diluting for 4a: we add Divide your cell density: 0.

And for 4b: we add Dive your cell density: 0. In In 20mL of 4b, there are: 0. The total volume being transfered is Thank you so much!!My research focused on mathematical modeling of the cell cycle in leukemia and involved experiments with cell lines.

During that time, I had to count cells with a hemocytometer so often to track growth that I got tired and decided to build an app, HemocyTapand share my knowledge on the topic here to help as many people as possible.

To calculate the dilution factor, you need two things: the original volume of the solution you dilute and the final volume after diluting or the volume you have added to dilute, in which case the final volume will be the original volume plus the volume you have added. Choose a specific notation from the dropdown box to change it to the standard one you use. See my other post if you need more help carrying out dilutions. This is assuming 45mL is your initial volume and 5L is your final volume.

If 5L is the volume you add, then you would put 5. Remember to change the units in the dropdown too. Are you sure those are the volumes, or are you sure 1. Can you give me a link to the instructions you are following to prepare this solution? I might be able to help further then. Hi I start with the dilution 0. So for instance, if you have 10mL, the first dilution would require you to add There needs to be a centrifugation step in-between or something like that to allow you to concentrate more.

If it was the other way round, e. Lets say I have dilution factors from 50 to Does that mean that I take e. Or do I do them separately by taking 1ml sample, 49 ml water for 50 dilution factor and then on a new tube with 2.

### Dilution Factor Calculator (Dilutant to Stock Ratio)

Hi Maria Fuentes, Nice website. Happy to visit this site. I have problem calculating these numbers. I have 10 ml of stock solution. Can you simplify this and tell me the step wise calculation so that it can be used in future.

Thank you. From the stock solution, get the amount of cells needed. You have to concentrate it, this is done by centrifuging the 3, Good evening Maria, Thanks for the calculator. I need your explanation for something still though.