Hello,
I am using the costar gel loading tips round 4853 and an eppendorf pipette (100 µl) to load my gels. The volume of my samples is 25 µl. Before loading, I usually set my eppendorf pipette to 25 µl and use the gel loading tips as mentioned above. But everytime I get air bubbles in my tips that result non-optimal western blots.
Do you have any tips/ideas how to avoid air bubbles in the tips?
What I've already tried:
I resuspend the tip to remove the air bubbles but in most cases the resuspending process results in more bubbles.
I pull the pipette slowly to avoid capture any air, but still isn't working.
I readjusted the volume of the pipette to 23,5 µl to set a lower volume to avoid sucking excess air, but then I lose sample since a small amount is still in the tube. So that doesn't work either.
I am thankful for any help and tips!
Because samples containing detergent tends to stick to the inside of the pipet tip, if you set the pipettor volume equal to the sample volume, you will always expel air at the end of the dispense.
Also, if the samples were heated, some of the water will have evaporated and be located at the top of the tube, lowering the sample volume. Centrifuge the samples after heating and allowing them to cool to room temperature to return the evaporated water to the sample.
Set the pipettor volume to a few microliters less than the sample volume to allow for the small portion of the sample that remains stuck to the pipet tip. Release the sample slowly into the well to allow time for most of the sample to drain off the sides of the pipet tip. Do not push the plunger to the bottom stop - stop pushing out the sample when the last of it has left the pipet tip.
This approach is easier when using a 20-µl pipettor than a 100-µl pipettor because the spring on a 20-µl pipettor plunger is not as strong. Reducing the sample volume from 25 µl to 20 µl will allow you to use a 20-µl pipettor to dispense the samples into the wells, and this will give you greater control over the dispensing.
Finally, if you allow the sample to fall into the well slowly from above, due to its greater density than the buffer (because of the glycerol), instead of placing the pipet tip near the bottom of the well, if a bubble if air is accidentally dispensed, it will not disturb the already-dispensed sample very much and will just float to the top without causing any trouble.
After all the effort that went into making the samples in the first place, spending a little extra time loading them onto the gel carefully is worthwhile.
@Adam B Shapiro Thank you for your comprehensive guidance! I'll proceed with my experiments based on your suggestions.
Furthermore, I have another question. If I adjust the pipettor volume to, for instance, 20 µl instead of the 25 µl you mentioned, I assume about 5 µl of the sample would be left in the tip. Is this not an issue? I'm concerned about consistently leaving behind a portion of my sample and how this might influence my experiment outcomes.
Dear Shang Chi
You can also try the following trick:
You set a pipette at 25 uL, load a sample into a tip, and then slowly adjust the volume on a pipette (with a sample still in the tip) until you remove air from it.
The other way is to set a pipette at, e.g., 20 uL, load a sample into a tip, and while keeping the tip in the sample solution, adjust the volume to higher values as far as you upload the whole sample into it.
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