Micropipetting SOP

A micropipettor is an expensive instrument that delivers a very accurate volume in the hands of a skilled careful operator, so pay attention while using these devices.

BIG NO-NOs:

--Do NOT use without putting a tip on the pipettor; the barrel should never touch the liquid, only the tip.

-Do NOT lay down the pipettor when its tip contains liquid. Keep it vertical!

-Do NOT attempt to set the volume above or below the stated range of the device. Pay careful attention to the volume scale and the range!

-Do NOT let the plunger snap back after taking up or ejecting liquid – this damages the piston. Maintain steady control of the plunger speed.

1. Examine your set of micropipettors to see what range of volumes is present in your set. Locate the plunger on top, the volume readout window, and volume adjustor, and tip ejector mechanism.

2. Insert a tip onto the shaft of the pipettor.

1. Get the correct tip to fit your pipettor. Color coding may help but is not always present (e.g. yellow tip to pipettor with yellow top).

2. Press firmly to create an airtight seal between the barrel of the shaft and the tip so the instrument can do its work in drawing up the precise volume of liquid.

Never insert a pipettor into liquid without a tip – this would ruin the piston that measures the precise volume.

3. Check the plunger stops on your instrument.

1. Place your thumb on the plunger. Use the rest of your fingers to grip the instrument with your index finger resting in the slot.

2. Gently, with steady pressure, depress the plunger till you reach the “first stop,” that is, till you can detect resistance. Then depress further, till you get to the “second stop.”

3. Repeat a few times to get the feel of the stops and to practice steady control of plunger speed.

Never let the plunger “snap back” because this can damage the piston.

4. Setting the Volume. These pipettors are termed “variable volume” because you can set the volume to a value within the range of the instrument. Each pipettor has its particular range of values. Examine your pipettor set for the volume range of each pipettor:

Locate the volume adjustor: sometimes this is the plunger itself or sometimes it is a knob near the top of the pipettor.

Next, look at the volume readout window; different brands of pipettors have different approaches to the readout scale. Your pipettor may have a direct digital readout with the decimal place clearly indicated for ease of use or it may have a micrometer readout setting that varies with the size of the pipettor (the Daiggers, for example). A direct readout is easy, no need to explain, the decimal points are shown. But the micrometer readouts require more attention. See appendix A for detailed directions if you are using one of these pipettor sets; tricky! Now set the desired volume on the correct pipettor.

Do not attempt to set the volume beyond the pipette’s minimum or maximum value – doing so damages the gears.

5. Working with Sample Liquid.

· Arrange your materials on your workspace for easy access, and sit at a height so that you can rest your elbow on the work surface.

· Pick up a micropipettor with a tip in place.

· Obtain a microcentrifuge tube containing sample. Hold the opened tube firmly in one hand at nearly eye level to observe the movement of the liquid as you operate the pipettor.

· Hold the micropipettor in a vertical position over the tube.

· To withdraw sample from the tube:

-depress the plunger to the first stop and hold. (This action displaces air equal to set volume from the tip – hence, the term an air-displacement pipettor.)

-Immerse the tip of the pipettor into the sample liquid. Do not push the tip into the very bottom of the tube as this may restrict withdrawal of the sample liquid.

-Slowly release the plunger to draw up the liquid into the tip.

-Pause for a second or two to allow full intake of liquid into tip.

-Remove tip from sample liquid. -Examine the liquid in the tip to be sure that there is no air space at the end of the tip. (There will be, of course, an air space on top where the tip meets the pipettor shaft.)

-If there is an air space, eject liquid sample and repeat sample uptake steps till there is no visible air space at the bottom of the tip.

-To assure maximum accuracy, you can examine the outside of the tip and wipe off liquid droplets that cling to outside, taking care to stay away from tip outlet to avoid removing liquid from the tip interior.

-Always keep the pipettor in an upright position while there is liquid in the tip, so that the liquid does not contaminate and damage the shaft of the pipettor.

· To deliver sample into a new tube:

-Insert the tip of the pipettor into the inside wall of the new tube. This creates a capillary effect to help remove sample completely from the tip.

-If you touch the tip into a liquid mixture, be sure to use a fresh tip for the next sample delivery to avoid cross-contamination of reagents.

-Slowly depress the plunger to the first stop, then on to the second stop to release the last bit of sample liquid. Pause for a second with plunger in depressed position to allow time for full release.

-Keep the plunger depressed while you remove the tip from the new tube out of the liquid (this avoids withdrawing liquid back into the tip!)

-Next, Release plunger gradually to resting position, and eject tip into discard container.