Is it normal to observe Partitioning Oil at the bottom of the pipette tip after collecting GEMs?

Question: Is it normal to observe Partitioning Oil at the bottom of the pipette tip after collecting GEMs?

Answer: Observing a small amount of Partitioning Oil (<15 ul) at the bottom of the pipette tip is normal and will not negatively impact assay performance. 

Note: If larger amounts of Partitioning Oil are observed (>15 ul), if the emulsion appears heterogeneous with variable GEM sizes, or if the total emulsion volume is reduced, a wetting failure and/or clog may have occurred. See the bottom of this article for further details.

Observing small amounts of Partitioning Oil (<15 ul) is normal. The appearance can vary based on the following factors:

1. Amount of time that elapses after GEM generation (ie. time that GEMs sit in the chip, pipette tip, and/or PCR strip tube).
2. Turbidity of Partitioning Oil.
3. Variation in emulsion retrieval (i.e. pipetting speed and angling of pipette tips).

Each of these factors is explained in more detail below. 

1) Amount of time that elapses after GEM generation

In all Chromium Single Cell assays, it is normal for some separation to occur between Partitioning Oil and GEMs after GEM generation, which results in a small amount of oil (< 15 ul) becoming more visible at the bottom of the pipette tip. This separation can occur over time if GEMs sit in the chip, pipette tip or PCR strip tube. As shown in the Figure below from a Next GEM assay, if GEMs sit in the pipette tip for ~1 minute, some separation occurs, and a small amount of oil is visible at the bottom of the pipette tip. This is normal and will not negatively impact assay performance.

2) Partitioning Oil turbidity

The turbidity/cloudiness and color of the Partitioning Oil varies between lots, as outlined in this article: Differences in partitioning oil appearance. This is normal and does not negatively impact assay performance. As shown in the Figure below, if clear Partitioning Oil is used, oil present at the bottom of the pipette tip or PCR strip tube may be easier to see; in contrast, if cloudy Partitioning Oil is used, oil present at the bottom of the pipette tip or PCR strip tube may not be as noticeable. 

3) Variation in emulsion retrieval

The speed and angle of the pipette tip may influence the amount of Partitioning Oil that is visible at the bottom of the pipette tip. Slower pipetting speeds may allow more time for oil to become visible at the bottom of the tip. Furthermore, if using a multichannel pipette, the angle of aspiration may impact the volume of oil in the pipette tip. This may lead to some samples having up to ~15 ul oil at the bottom of the tip, while other samples may have less oil. If >15 ul is observed, a wetting failure or clog may have occurred.

Wetting failures and clogs

If larger amounts of Partitioning Oil are observed (>15 ul), if the emulsion appears heterogeneous with variable GEM sizes, or if the total emulsion volume is reduced, a wetting failure and/or clog may have occurred. We recommend re-running a sample if a wetting failure or clog occurs. See the following articles for further details:

• Best Practices to Minimize Chromium Next GEM Chip Clogs and Wetting Failures
• How do I diagnose and report a clog failure with GEM-X assays?
• How do I diagnose and report a wetting failure with GEM-X assays?

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