The art of pipetting: Common errors, big blunders.

The art of pipetting: Common errors, big blunders.

Write By: admin Published In: Troubleshooting Created Date: 2016-09-09 Hits: 458 Comment: 0

Imagine this: You’ve obtained this standardized and highly cited protocol for this crucial gene. You’ve worked out all the requirements, gathered all the reagents and are all set to start with your very first step: plasmid extraction. You’ve followed the protocol through-and-through. And just when you think you have prepared the best possible plasmid, you end up with too much contamination. What could possibly have gone wrong?

The art of pipetting: Common errors, big blunders.

Imagine this: You’ve obtained this standardized and highly cited protocol for this crucial gene. You’ve worked out all the requirements, gathered all the reagents and are all set to start with your very first step: plasmid extraction. You’ve followed the protocol through-and-through. And just when you think you have prepared the best possible plasmid, you end up with too much contamination. What could possibly have gone wrong?

Of the many possible reasons, one of the most common mistakes include errors in pipetting. Yes, you heard that right! It might sound bizarre because at a stage when you’re capable of designing entire experiments it’s unlikely that you could be making such trivial errors! Think again.

Did you hold it right? Did you check whether the liquid was properly ejected? You didn’t jam the tips right into the tip box, did you? Were your tips autoclaved? Was your pipette clean and calibrated? Pipetting is no rocket science, but it does require precision while handling. The manufacturers have done their job to ensure high efficiency, it is now upto you to extract the cent percent. Here we list some tips to help you get it right.

·       Keep calm and keep clean: It all begins with cleanliness. Ensure that your pipette is clean: try to focus more on the ejector part as it is this region that comes in contact with your solutions (technically it shouldn’t, but you know!) Ensure that your tips are autoclaved.

·       Begin when you’re all set: The next rule is to set up everything: loosen the cap of your falcon tube, open the cap of your microcentrifuge tube and keep the booklet in front. Set the desired volume, open the tip box and bon voyage! Do not hold the pipette until you’ve ticked these off your checklist.

·       Mind your (tip’s) head: Yes we know how excited you are to get on with that experiment. But calm down fella because this is important. While installing tip, do not jam too much into the tip box. Take it easy. Damaging the tip head could lead to errors in dispensing/aspirating. Apply a consistent force to ensure perfect fit.

·       Get your tip used to it: A good practice is to pre-wet the tip with your solution. Just aspirate and dispense the liquid to get the inside surface of the tip uniformly coated with your solution. It’s like allowing your tip to get used to the new solution. It helps!

·       Take it easy, take it slow: You’ve been doing great so far. But still, things can go really wrong. Remember to dispense your solution at a steady pace. Avoid jerky movements. If you’re holding the pipette with your right hand, support the ejector with your left hand (forefinger?) to hold it steady. Stop dispensing when your ejector reaches the first stop (level 1) to avoid any air bubbles.

·       90-45 rule: Always remember to hold the pipette and aspirate at an angle of 45° but dispense at an angle of 90°. Do not hold it in inverted or horizontal position as the liquid can enter into the pipette (remember, rule 1?). For dispensing, aim right into the tube, support the pipette, keep it steady and dispense! Try to stay away from the walls of the tube because, keep-pipette-clean! When you’re required to dispense at the side, the tip may touch but be sure to keep the ejector away.

·       On the (sur)face of it: So you need 1µL. You set the pipette, get the tip and dip it right into the solution because 1 µL is hardly even visible. For you it is 1 µL, for the solution it is 3 µL. Can you figure out where you went wrong? When you dipped that tip into the solution, the tip contains your desired 1 µL but it is also coated on the outside with the same solution thus increasing the actual volume that is being added into your reaction mix. To avoid this, try to dip the tip just a little below the surface and aspirate. Now, even if you need to dispense by diving into the reaction mix, you’re good to go!

·       Buy good quality pipettes and tips: It is important to get hold of pipettes and tips from a reputed manufacturer. That’s like half the battle won. At labekart, you can avail top quality pipettes from Abdos and Eppendorf suited for all your needs. Good quality, sterile tips capable of accommodating a range of volumes are available from Abdos.

 

So, what are you waiting for?

 

 

Tags: micropipet pipet liquid handling

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