without a details explanation of what you are doing it is difficult to trouble shoot what is going wrong. There is a possibility that you are wrecking your droplets before they get loaded on the reader. This can happen if you are too rough when you pipet (or your pipet tips have too small of an orifice), or you are leaving them sit too long, or you have spun down your plate after loading the droplets. There are a number of other things that could lead to either ineffective droplet generation or droplet degradation after they are made, but with no indication of the protocol you are following it is difficult to say. For now, try a few samples and try to be very gentle with your droplets...don't jam your pipet tip against the bottom of the cassette as you withdraw them (keep your pipet at a 30-45 degree angle) and gently dispense them onto the wall of the pcr plate (the oil/sample mix will slide down the tube wall to the bottom of the plate). Also try using some pipet tips with a wider bore if yours appear to be tight. Never pipet your droplets more than once, they should go from the cassette to the plate that they will be PCRed and read in directly.

Good Luck

Ron

Hi, Thanks a lot for your reply. Although I saw it after I ran my samples again and luckily it worked well this time, but you are right and your tips make perfect sense. I appreciate your help.

I know this is way after the fact, but someone up voted this so I went to check out what it was! I noticed one very important thing that I missed here for others coming to read, and that is that the generation oil has an expiry date, and if you are using the Sybr chemistry the oil does actually go bad fairly soon after that expiry date. If your oil is looking milky even before droplets have been made then your oil is starting to go off. Using this oil will result in fewer and fewer droplets that will pass quality control, so if you are having issues with the number of droplets across a multitude of samples....check your oil!

Cheers

Ron

I am a begginer in this technology and I have many doubts

How about pipette tip?

Do you recommend any brand?

Thank you so much for your time Ron Reade.

Bio-Rad has some specific recommendations that you can look up, however we have used both the Axygen maximum recovery filter tip (20 and 200ul) and we have also used the Neptune brand. Having said that, I would not sweat the tip brand too much unless you are having consistent issues with droplet quality. I suspect that you will have some issues to start as it takes a bit of time to get used to the technique. What I find did make a big difference was getting a new set of pipets, the Gilson Pipetman L series is amazing, and are a huge upgrade from our previous versions. Very smooth easy pipetting: http://www.gilson.com/en/Pipette/Products/90.339/Default.aspx#.WP4p8_krJpg 

Good luck Francisco

Thank you Ron Reade. 

The Bio-Rad mentions that the number of droplets per reaction is 20k. However, it is not the real situation. Most of the time it never reaches 20k with an average of ~15k (12k-19k). Now coming back to the question of getting very low counts like less than 10K, there are many good suggestions by Ron Reade above. I want to summarize and add a few additional points.

1) Be gentle with droplets if you are using a manual droplet generator. The handling is more optimized if you use automated droplet generator.

2) As Ron Reade mentioned above that try to take everything at once and not pipette multiple times. Try to be consistent and if you change the way you pipette out then the amount of droplet may change. As bio-rad suggests taking out 40ul so you should take out exactly that amount in one go.

3) Bending pipette at an angle as mentioned above is good as it helps in easy movements of droplets into the pipette tips.

4) Keep patience while pipetting out the droplets into 96-well plate. Droplets take some time to slide down. Just go till the first lock while pipetting out and wait for some time till more liquid slide down and then push out the sample by pressing to the second lock that will release everything into the well. Wait for some time after the last push as droplets may take some time to slide down with your last push pressure.

5) Be careful while taking your plate out of the thermocycler. Some regions may have static current and if you don't ground yourself, it may break the droplets. So touch any metallic surface before taking your plate out of the thermocycler.

6) Don't take out the plate immediately after PCR is done. Once PCR is over the sample temperature comes down to 4 degree immediately but the top cap temperature remains 105 degree. If you take out the plate immediately then there is a sudden change in the temperature that may also not be good for the health of droplets. So wait for about 15-20 minutes before taking out your plate out of thermocycler.

7) If the sample is on ice before making the droplets, it may have higher viscosity. If sample is at room temperature then that is good for the best droplet generation. So after you make your reactions it may be a good idea to keep the reactions at room temperature for 10 min before making droplets. The automated droplet generator manual also suggest keeping reaction at room temperature for 10 min before generating droplets.

8) Bio-Rad has come up with a new machine where droplet generation, thermocycler, and droplet reader are in one box. That machine is more consistent in terms of generating an equal (and high) number of droplets everytime.

Good luck with your droplet generation.