1. Using a Micropipette Huntington Gardens July 2013
MOLECULAR BIOLOGY - THE STRUCTURE OF DNA (INTRO)
GENETIC ENGINEERING AND WORKING WITH DNA (INTRO)
MICROPIPETTING (LAB1)
Using a Micropipette Huntington Gardens July 2013

2. Micropipette Use Twist dial to desired volume
Add disposable pipette tip
Press plunger to first stop
Insert pipette tip into solution to be
transferred
Slowly release plunger to retrieve liquid
Move pipette tip into desired tube
Press plunger past first stop to second stop
to transfer liquid, keep the plunger down
as you remove it from the tube.
8. Eject tip

3. Micropipette Use 1. Twist dial to desired volume
. Add disposable pipette tip
. Press plunger to first stop
. Insert pipette tip into solution to be
transferred
. Slowly release plunger to retrieve liquid
. Move pipette tip into desired tube
. Press plunger past first stop to second stop
to transfer liquid, watch liquid stick to wall
of tube. Remove tip, then release plunger.
8. Eject tip

4. Reading a Pipette
P-20
For Lab 1: Only set out P-20’s
Pipette Limits:
Never below 2.0 µL
Never above 20 µL
5.0 µL

5. Reading a Pipette P-200 P-1000 P-20 5.0 µL 50 µL 500 µL
Different pipette sizes measure different amounts even thought the window on the pipette reads the same (“0-5-0” in this example)
5.0 µL
50 µL
500 µL

6. Two pipettes: one set to 20µL, one to 2µL
Notice at 20µL the plunger is much higher so you will push a lot farther than with 2µL, which needs hardly any pressing to reach the first stop.
Two pipettes: one set to 20µL, one to 2µL

7. Pipette Practice Word Doc

8. Teacher Preparation: Mixing the Buffer Solution for Lab 1:
Comes as 20x (or 10X)
Dilute with dH2O.
1X is the working solution to be used for the labs.
You will need buffer for electrophoresis:
To make your ‘gels’
To fill your electrophoresis box

9. -Want XmL of 1X Buffer -Have 20X Buffer
C1V1 = C2V2 (c=concentration, v=volume)
C1= desired concentration =1X
V1= desired amount = .5L = 500mL
C2= stock solution (what you have) = 20X
V2 = Unknown (solve for)
Equation: (1X)(500mL) = (20X)(?mL)
?mL = 25ml
Bring to volume with 475 mL dH2O

10. Making a 0.8% Agarose Gel
Estimate that each gel tray holds 25mL of agarose gel.
1mL = 1gram
Equation as percentage (cross multiply)
0.8% of 30mL is 0.24 grams of agarose in 30 mL of 1X Buffer.
.08 x 30mL = 0.24 grams agarose

11. Making a 0.8% Agarose Gel
1)Estimate that each gel tray holds 25mL of agarose gel.
6 gels = 180 mL (I round to 200mL)
.08 x 200mL = 1.6 grams agarose
A 0.8% gel for a volume of 200mL is :
1.6 grams of agarose in 200 mL of 1X Buffer.
2) Heat in microwave until all “flecks” are dissolved.
3) When cool enough to touch, pour into combed gel tray.

12. Tips:
If you’d like to let students pour the gel you can keep melted gel in falcon tubes in a hot water bath around 60°C.
(Keep in mind, this is after you melt it in an Erlenmeyer flask, in the microwave at a boil)
A hot water bath is not hot enough to dissolve agarose.

13. Lab 1: An Introduction to Microvolumetrics and Pipetting
Please read through and complete Lab 1
Keep in mind the directions as you read to see what vocabulary may be difficult for your students.
Lab 1: An Introduction to Microvolumetrics and Pipetting

14. Loading Gels: Insert pipette tip: Under buffer level Above gel well
PIPETTING LIQUID INTO THE CENTRIFUGE TUBE (LAB 1)
LOADING A GEL (LAB 1)

15. RUNNING A GEL

16. Micropipette tip should be ABOVE the well NOT IN IT!!!!

17. NICE!
Pipette tip is under the buffer level, but above the gel

18. Lab 1 Conclusion Questions:

19. Magnified image of Agarose Matrix:

20. Lab 1 Conclusion Questions:
You should have guessed that Xylene Cyanol was the largest from the results, but the Bromophenol Blue has Br groups that make it very negatively charged so it is “pulled on” harder by the + electrode

21. Transformation
TRANSFORMING BACTERIA (LABS 2A & 4A)