I was so excited to start using 384-well plates for my assays. With so many wells, these plates are useful for testing many conditions in parallel, as required in ELISAs, siRNA library screens, and drug treatment dilutions. However, I quickly learned that pipetting in these plates is more complicated than I thought. This article contains some practical hints to help you handle this plate format.
While some labs may be able to handle this job automatically with a pipetting robot, yours may not have access to one. Additionally, sometimes it is more work to program a pipetting robot than it is to accomplish the job manually.
Getting in the Mindset
These are the things to keep in mind before you start.
- When booking time in the biosafety cabinet or planning your day, over-estimate the length of time you need. Tackling pipetting jobs of this scale invariably takes longer than you expect.
- If your lab allows music, listen to tunes that enhance your focus, sans lyrics. I find the white noise of smooth jazz to be very soothing. Others prefer classical music for this purpose. Avoid songs with lyrics if you’re prone to singing along, as recalling the words diverts attention from the task at hand. For the same reason, also refrain from listening to podcasts or having conversations.
- Choose and double-check your pipette equipment. Accuracy becomes more important in 384-well plates because there is generally less overall volume. Make sure you choose the most accurate pipette size for your pipetting volume. For example, to dispense 3 µL, choose a 10 µL pipette instead of a 20 µL pipette. If you are using a multi-channel pipette, be aware that the tips may be spaced for compatibility with 96-well plates. Therefore, in the 384-well format, multi-channel pipettes dispense into every other well.
- Lay out every step of the experiment in a program like Microsoft Excel and take printouts with you to the bench. Second-guessing yourself during a pipetting experiment is the worst-case scenario. The likelihood of doubt decreases with the more effort you invest in planning.
Keeping Your Place on the Plate
A 384-well plate is a 16×24 matrix, which is too much for the average human brain to handle. Wells near the periphery of the plate are easy to spot quickly, but it becomes more difficult in the middle of the plate, which can really slow you down if you have to count every time.
- Sub-divide the 384-well plate into manageable sections Use a small piece of tape or sharpie marks to subdivide the plate. For example, I find that dividing into 16 sections, each containing a 4×6 array of wells works great (see title picture above).
- Use pipette tips methodically, corresponding to the target well. For example, if you’re pipetting into well E8, choose the pipette tip in position E8 on the pipette tip magazine. Match the markings of your plate on the micropipette magazine. Note that pipette tips come in standardized magazines of 96, so each reagent you pipette into a 384-well plate will use 4 magazines.
- Audibly say the coordinates of each target well. Don’t be self-conscious about others hearing you talking to yourself. If they’ve lost their place while pipetting, they’ll understand.
Disarming Technical Difficulties
These tips are helpful to navigate the subtle difficulties associated with so many wells and so little volume.
- Change your gloves about every 45 minutes. Sweaty hands are uncomfortable while pipetting. Apply baby powder between glove changes to absorb moisture.
- Gently touch the bottom or side of the well to dispense the solution. If you are pipetting small volumes, sometimes surface tension can prevent proper dispensing of the liquid.
- Keep the plate tilted to double check which wells have solution in them. If you work in a fume hood or biosafety cabinet, you cannot look directly downward into the plate because of the sash. Propping up the back of your plate can help you visually monitor your status.