Cell Biology Lab

Pipetting Directions

• Blue pipettes (Gilson/Rainin)- turn wheel.
• Black pipettes (Eppendorf)- turn dial.
• Gray pipettes (Eppendorf)- pull end out and turn.

There are four models of the Eppendorf brand pipettes.

• 0.5-10ul Clear tips
• 2-10ul Yellow tips
• 10-100ul Yellow tips
• 100-1000ul Blue tips

Do not overturn any of these as you will damage them.

Use of micropipettors.

Nevers

• Never rotate volume adjustor beyond the upper or lower range.
• Never use without a tip in place.
• Never lay pipettor down with filled tip-fluid could run back into the piston.
• Never let plunger snap back after withdrawing or ejecting fluid. This could damage the piston.
• Never immerse anything other than the disposable tip in liquid.
• Never flame the micropipettor tip.

1. Rotate volume adjustor to desired setting. Note change in plunger length as volume is changed. Be sure to locate decimal point correctly when reading the volume setting.

2. Firmly seat the proper-sized tip on end of micropipettor

3. When withdrawing or expelling fluid, always hold the tube firmly between thumb and forefinger. Hold close to eye level to observe change in fluid level in tip. Do not pipet with the tube in rack or when someone else is holding the tube.

4. Hold the tube by the body, not by the lid, and hold the pipettor almost vertical.

5. Most digital micropipettors have a two-stop position plunger. Depressing to the first stop measures the desired volume. Depressing to the second stop introduces an additional volume of air to blow out any remaining fluid from the tip.

6. To withdraw fluid from tube.

a. Depress plunger to first stop and hold. Dip tip into fluid and gently release thumb.

b. Slide pipette tip out along wall of tube to dislodge remaining droplets adhering to the outside of the tip.

c. Check that there is no air space at the very end of the tip. Learn the approximate levels that particular volumes fill the tip.

7. To expel sample into reaction tube

a. Touch tip to wall of tube

b. Slowly depress plunger to first, and the to the second stop to expel fluid.

c. While keeping the plunger at the second stop, slide the tip out of the fluid, along the tube wall and out of the tube.

d. Eject the tip into trash.

8. To prevent cross contamination of reagents.

a. Use a fresh tip for each new reagent to be pipetted.

b. If tip become contaminated, replace it with a new one

Small volume exercise

This exercise simulates setting up a reaction with a micropipettor in the range of 0.5-10ul or 1-20ul

1. Use marker to mark three- 1.5ml tubes A, B, and C.

2. Use matrix below as a checklist while adding solutions to each tube. All volumes are in microliters

Tube	Solution 1	Solution 2	Solution 3	Solution 4
A	4	5	1	0
B	4	5	0	1
C	4	4	1	1

3. Add each solution to the appropriate tubes, being careful to avoid contamination. Sharply tap the tubes to pool the samples at the bottom.

4. 10ul should have been added to each tube. Set a pipet to 10ul and carefully withdraw all of the fluid into a fresh tip.

• Is the tip filled? or
• Is a small volume of liquid left in the tube? or
• Is an air space present in the end of the tip? The actual volume can be determined by rotating the dial until no air remains in the tip, and then reading the volume directly

Large volume exercise

This exercise uses larger volume pipettors. It is easier to make errors with these larger pipettors if the plungers are not depressed and released slowly.

1. Mark two tubes E and F. All volumes are in microliters.

Tube	Solution 1	Solution 2	Solution 3	Solution 4
E	100	200	150	550
F	150	250	350	250

2. 1000ul (1ml) should have been added to each tube. Set a pipet to 1000ul and carefully withdraw all of the fluid into a fresh tip.

• Is the tip filled? or
• Is a small volume of liquid left in the tube? or
• Is an air space present in the end of the tip? The actual volume can be determined by rotating the dial until no air remains in the tip, and then reading the volume directly.