- Applications - General
- Applications – RNA-Seq
- Product demonstration/workshops
- Purchasing, installation and post-sale support
- Dolomite Bio
- Dolomite Bio
Applications - General1.1 Will my cells work with your systems?
In most cases, yes, your cells will work with the system. Whether a cell is compatible or not is dependent on many things such as the size and fragility of the cells. Please contact our applications team for a detailed list of cells that have been trialled in the RNA-Seq system.
The droplet junction size of the chip tends to dictate the size cells that can enter the system. We advise that cells are half the size of the junction, to avoid any sheering of cells and blockages.
The µEncapsulator1 system allows for samples of cells as small as 100 µL.
Cells and beads are advised to be used at a concentration of 300 per µl.
Currently the most practical way of doing this is making small double emulsion droplets (an aqueous droplet in an oil shell, with an aqueous continuous phase), that can be sorted on a conventional FACS (https://www.dolomite-bio.com/applications/droplets-facs-sorting-libraries/).
To get this out, you would have had to put the chip in a beaker of boiling water and then flush the chip through.
Yes, please see the “Applications” tabs of the specific Product pages.
At present, we do not sell beads.
Yes, we sell the Picosurf™ range of products.
- Pico-Surf™ 1 (In Novec7500)- Great for PCR. Stable, chemically inert droplets for PCR.
- BioRad Droplet Generation Oil For EvaGreen – Suitable for RNA-Seq applications
Pico-Surf™ surfactants are biocompatible specialized chemical solutions. They contain a proprietary surfactant in a fluorocarbon carrier oil and are ideal for generation of robust and stable droplets. The surfactant acts as an interface stabilization agent that enables stability of the droplets under a wide range of temperature and biological conditions as well as allowing droplets to be stored for several weeks in suitable conditions.
Pico-Surf 1 5% in Novec 7500 has been successfully used with:
• Mouse hybridoma
• Mouse B-cells
• Human B-cells
• Human T-cells
• Jurkat cells
This is because different types of beads are used in each application. RNA-Seq requires beads which are barcoded and at present are a size of 30-50µm. These beads therefore only run in a 100µm chip such as the RNA-Seq chip. B and T cell studies require beads which don’t have a barcode and therefore are a size of 10-20µm, which are able to run in the µEncapsulator 30/50µm chip.
Typically 20% larger or smaller than the junction size (this is achieved by varying the pump pressure/flow rate).
For the RNA-Seq system we make droplets around 88um and for the uEncapsulator we typically make 50um droplets (using a 50um chip). This can be adjusted, as some customers may have a small bead and small cell, so the size of the droplet can be decreased, using a different size chip.
Around a week at room temperature.
No, the Dolomite Bio systems provide a solution for encapsulating single cells into droplets with/without beads. The protocol ends with a set of beads with mRNA attached, ready for downstream processing.
Yes, the system is flexible depending on your application
Applications – RNA-Seq2.1 What is the recommended concentrations of beads/cells in the RNA-Seq system?
The concentration we advise (300 per µl) reduces the chance of doublets of either a cell or bead. The system can be manipulated to higher concentrations to increase the chance of 1 bead and cell per droplet.
This is dependent on the concentration of the beads and cells you are using, but when using our advised concentrations, the capture efficiency is 1/20 for both beads and cells. Meaning that 1/400 droplets contains a bead and a cell.
- A sample loop is used in the RNA-Seq system to efficiently flow beads into the chip. It is a 10m length of 0.25mm internal diameter tubing, which is coiled together into a loop, and has a total internal volume of 500 µL. The idea of this is to ensure that the beads are evenly distributed inside the sample loop, and pressure can then be applied to sweep the beads along, into the droplet junction.
- This method minimises blockages that are caused by the current beads used by Macosko et al. The beads are also easily damaged, very dense and very sticky, so the use of a sample loop prevents any harsh agitation that can cause damage to the beads and separates them properly, to avoid doublets.
- It allows for an even distribution and flow of beads throughout the run, whereas when using other methods of injection, due to the high density of the beads, they are very prone to settling out of solution, clumping, and therefore not flowing well through a microfluidic system.
The beads are prepared at the correct concentration and then loaded into a 1ml luer lok syringe. The beads are loaded into the loop via a luer lok syringe fitting. It is very important to make sure the beads do not settle to the bottom of the syringe while loading, so tilting back and forth by 180 degree movements of the syringe while loading, prevents this.
This is due to two main reasons:
1) The junction size at present on the µEncapsulator. The RNA-Seq system uses a 100µm chip, and the beads mentioned in the Macosko et al paper. These beads vary in size between 30-50µm, so are compatible with the RNA-Seq chip as their size is able to flow through the junction. However, the µEncapsulator has a junction size of either 15µm, 30µm or 50µm. This means that the beads are the size of the largest junction size, and wouldn’t flow through, without blocking.
2) The density of the beads used in the RNA Seq process is relatively high and therefore the use of the sample loop described above is the most appropriate method of introduction (rather than loading into the µEncapsulator Reservoir Chip).
25,200 per hour (at what concentration does this occur? 1/20 for cells and beads and at the highest recommended flow rates)
Product demonstration/workshops3.1 How can I see your products?
If you are looking to see the products live and running, we regularly run workshops in many countries around the world.
Keep up-to-date with workshops being run near you by visiting the Workshops page.
Purchasing, installation and post-sale support4.1 Do you provide installation and training?
Yes, installation and training can be (and typically for all system purchases, will be) included in the quotation. Please check your quotation.
Installation and training involves a specialist from the Dolomite Bio team visiting your institute to firstly install (set up and test performance of) the system. It also includes dedicated training of you and your colleagues, where we can help you understand the system, how to set it up and give guidance on your application. This often proves essential for users who have not used a microfluidic platform or are unfamiliar with the application and its set up.
If you run into any problems with the system, please contact our support team at firstname.lastname@example.org
Products5.1 Are the chips consumables?
If taken care of using the cleaning protocol after every use, the chips can be used and reused many times.
Normally the chips and system are cleaned by flushing through with 1% detergent, acetone and then air (to dry the system/chip).
We are able to advise you and produce system quotes on products if you are unsure of what you need to run your application. Please contact us.
We don’t recommend it as there are typically integration problems and issues with inferior pumps.
We will not be able to offer support for these types of systems.
Dolomite Bio6.1 What is the difference between Dolomite and Dolomite Bio?
Dolomite provides microfluidic components, such as pumps, chips and other accessories, which can be used for various different applications or function as microfluidic system solutions.
Dolomite Bio is focused on developing and selling products, typically complete systems and consumables to biologists. Many of these products are focussed on specific biological technologies such as Single Cell RNA Seq. Some of these products contain microfluidics but this is not the focus of Dolomite Bio.
Yes, vacancies are updated regularly on our Careers