DON'T dilute the 2x component to 1x (in this case and in many other cases) with water and store it back to -20C. You store the 2x solution.

To make it final concentration of 1x, depending on the final volume of your reaction. If your final volume of the reaction is 20 ul, then add 10 ul of the 2x [ddPCR Supermix for Probes (No dUTP)] to the reaction tube with other components. So the final concentration of the [ddPCR Supermix for Probes (No dUTP)] in it will be: (2x) x 10/20 = 1x.

Please refer to a similar example (Table 2. Preparation of the reaction mix) from the attached datasheet in my previous answer.

The info should appear on the product datasheet which comes with the kit.

See this one from Bio-Rad (ddPCR™ Copy Number Variation Assays): "Storage and Stability-- ddPCR assays are stable for 12 months when stored at 4°C protected from light. The 20x assay mix can be kept at –20°C for long-term storage."

Thanks alot Yuan Yeu Yau

Dear Yuan Yeu Yau,

I want to ask concerning on the 2x ddPCR Supermix for Probes

(No dUTP); it should be diluted with dH20 to achieve 1x. Is it after i dilute with 4ml of dH20, i still can kept at 4degreeC?

DON'T dilute the 2x component to 1x (in this case and in many other cases) with water and store it back to -20C. You store the 2x solution.

To make it final concentration of 1x, depending on the final volume of your reaction. If your final volume of the reaction is 20 ul, then add 10 ul of the 2x [ddPCR Supermix for Probes (No dUTP)] to the reaction tube with other components. So the final concentration of the [ddPCR Supermix for Probes (No dUTP)] in it will be: (2x) x 10/20 = 1x.

Please refer to a similar example (Table 2. Preparation of the reaction mix) from the attached datasheet in my previous answer.

Dear Siti Radziah,

As we do in my lab, I recommend you to aliquot your master mix and conserve it at -20°C: when you thaw it first time, you separate the remaining volume by small volumes (like 88uL: 1x 8 samples mixes+ pipette dead volume) in small tubes (200uL).

Like this you only thaw twice your mix but you can conserve it longer at -20°C !

We froze everything in aliquots at -20°C

a) ddPCR Supermix for probes (NO dUTP). (one aliquots for 10 reactions)

b) Taq Man Copy Number Reference Assay,

c) Taq Man Copy Number Assay.

Our suggestion is that b) and c) can be frozen and thawed by several cycles and it is better to use a) always without cycles.

Hopefully it helps.

Thanks alot Yuan Yeu Yau Bingqian Qu, Leonie Goeminne,

How long to thaw the components?

What is the rpm,Celsius and duration to centrifuge?

Hi Siti,

I think you are asking a question about 'Reaction Setup' step in the protocol of ddPCR™ Copy Number Variation Assays (see attachment):

"2. Thaw all components to room temperature. Mix thoroughly by vortexing the tube to ensure homogeneity because a concentration gradient may form during –20°C storage. Centrifuge briefly to collect contents at the bottom of each tube and store protected from light."

• If this is the case, since it asks to 'thaw the components to room temperature', then just thaw it and let it sit at room temp. Check/feel it with your fingers. Use them when you don't feel they are cold anymore.
• The centrifugation step is just to collect contents on the tube walls after vortexing. So after you thaw them to room temp, vortex them, then just briefly centrifuge it at room temperature. We use a simple Mini-centrifuge to do this kind of job on bench (see attached figure, it reaches its top speed at 6,000 rpm in seconds). You can also use a regular centrifuge to do the same job. [http://www.universalmedicalinc.com/benchmark-mini-centrifuge-with-2-rotors.html]

What does mean No Call in my ddPCR reaction? Any ideas? droplet digital PCR/QuantaSoft software?

Below are the Component that I used in my ddPCR reaction

Water 5uL

MasterMix  12.5uL

20x FAM  1.25uL

20x VIC  1.25uL

digested DNA 6.6 ng/uL 5uL

It means that the software was not able to put the threshold. You can do do it manually if you can distinguish positive and negative droplets. If not, something went wrong with your PCR. Is it you first trial ? Do you have a picture of your graph ?

Here is the picture.

Actually this is my second trial. Still getting the same result

In my opinion, you have put to much references. All your droplets are positives on the HEX channel and you have no signal in your FAM channel. I'm not familiar with copy variation assay but it seems that you should reduce the amount of dna.

Have you try with absolute quantification (abs) at first to see your absolute FAM signal ?

Thank you so much Leonie Goeminne. I have try recuded the amount of DNA its work but then some well still display NO CALL status.

Anyone know what is the minimum concentration (copies/uL) that should obtained?

in ABS experiments, 1 positive droplets correspond to 0,04 to 0,09 c/uL depending of the total of droplet analyzed.

@Siti Radziah

We can get good quantification from around 37 copies per reaction to 131,000 copies per reaction with low uncertainty (see attached). IMHO I don't think that you are under the limit of detection for ddPCR but rather that the reaction isn't working.

Similarly, you are not over the saturation point (which is > 140,000 copies per reaction) because of the fluorescence intensity of your droplets. An intensity of around 2000-3000 will be negative droplets, not positive droplets (the positive droplet population will probably be at around 10,000 for this assay) so you definitely don't have complete saturation of your droplets.

Based on the baseline shifts (the change in negative droplet fluorescence between wells) it looks like you are not mixing your reaction mix sufficiently after you combine the mastermix, primers, probes and DNA solution. Optimally there should be less than 5% difference between the average fluorescence intensity of the negative droplet populations between wells, in the images you present above the variability is much higher than that. If your reaction solution is not homogenous before you dispense into the droplet generation cartridge then your droplets will not contain the reagents in the proper concentrations and PCR will not proceed.

Try vortexing your reaction mix for 10 seconds prior to dispensing onto the droplet generation cartridge. Based on your images, you should have negative droplet fluorescence at around 2800 in your VIC channel if you vortex first.

The negative droplet populations are pretty tight within each well so it looks like the droplet formation worked well and you have not been mis-handling the droplets (being rough with the emulsion or pipetting it too quickly when transferring into the 96-well plate).

Article Evaluation of a Droplet Digital Polymerase Chain Reaction Fo...

Dear Jacob L H. Mclaughlin,

Thank you so much for your earlier feedback. Which mean it is not acceptable if my  concentration(copies/uL) as we can see at below attachment below 37?

The intensity of around 2000-3000 will be negative droplets, you reflecting the column of negative as attached below. Am i right? But as you can see on my data that I send to you shows that the intensity is higher than the range you mentioned. So, it is not accepted is it?

Whereas for positive droplets, is below than the range you mentioned. 

Please assist me. Thanks alot.

The column of negatives in the table is the total number of negatives. Further across there is a column called MeanAmplitudeOfNegatives, which is the one that I am referring to.

It is also the y-axis in the 1D Amplitude plot.

Thanks for your quick replay Jacob L. H.Mclaughlin.

as you can see in my data the mean amplitude of negative shows the intensity -1225,... and  some well display over than the value of 3000. So what I need to do,in order to achieve the optimum range of intensity? 

And for mean amplitude of positive , what is the minimum range? is it ok less than 10000.