We are currently doing ddPCR tests, but we have droplets generation problems (not enough droplets...). Did someone already encounter this problem? Do you have any advice for us? Thank you!
Hi Célia,
This is a common problem so you're not alone! I have a lot of experience using the Bio-Rad QX200 and have found/been advised several things to maximise your droplet numbers:
1) Careful and smooth pipetting when transferring droplets (this is probably the most important). Over agitating can cause a breakdown of the droplets,
2) Ensure you digest your DNA if using amounts greater than 60 ng. This helps evenly partition the DNA, but also if you have too much high molecular DNA this can block the capillaries, preventing droplet formation,
3) When transferring droplets ensure the pipette tips is angled (ie not flat to the bottom of the cartridge) as this can cause a vacuum and sheer forces that can damage droplets,
4) I've found that if droplets are left to sit for more than an hour before PCR then a reduction in droplet numbers is seen. So if it's taking you a long time to generate and transfer droplets reduce the number of samples you run at any given time. It's better to perform more runs but get good results, than lots of reactions at once with low droplet numbers,
5) Consider your plasticware. When I was starting out with ddPCR I compared several different 96-well plate manufacturers, for whatever reason (maybe residues left on the plastic, or smoothness of the well walls) I found Eppendorf twin.tec 96 well plates maintained the best droplet numbers.
6) As with point 5, the same goes for pipette tips, I have found Rainin to be the best for this.
7) If you're not already consider using a multichannel pipette to transfer droplets. The plunger length is important to consider as it helps maintain the smooth movement. Most new ergonomical pipettes have very short plungers as they are usually easier to use, but for this you want a longer one. This is the pipette I use ( https://www.anachem.co.uk/Pipettes/Multichannel-Manual-Pipettes/Pipet-Lite-XLS%2B/Pipet-Lite-Multi-Pipette-L8-50XLS%2B/p/17013804 ). Bear in mind, some multichannels (this one included) need specific tips.
8) This a very expensive option, so only if you're going to be doing ddPCR in the long term, if using the Bio-Rad platform consider the autoDG. This is an automated droplet generator. This generates and transfers the droplets, so no manual handling is needed,
9) Finally practice transferring the droplets.
Hope some of that helps!
Adam
Thanks you for your help! we will take into account your advice and especially practice!
Célia.
Our lab has the automated DG (BioRad) and past few days have also been having problems.. either no droplets generated or very low numbers!!
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