1. An Introduction to Microvolumetrics and Pipetting
Laboratory 1

2. Overview Purpose: Familiarize students with small volumes
Introduce proper use of pipettes
Practice loading and running gels

3. Introduction Working with extremely small quantities Why necessary?
Very high cost of purifying enzymes and DNA
Hence, need to use instruments to handle small quantities
Volumes measured in:
Microliters (μL)
1 μL = mL
10 μL = 0.01 mL
100 μL = 0.1 mL
1000 μL = 1 mL

4. Materials Reagent Equipment & Supplies Solution 1 Solution 2
Distilled water (dH2O)
0.8% Agarose gel
1 X SB
Equipment & Supplies
1.5 mL microfuge tubes
P-20 micropipettor (2-20 μL)
Disposable pipette tips
Marker
Electrophoresis equipment
Power supply
Plastic microfuge tube rack

5. Materials Kim wipes Disposable bags Spatula Gloves Styrofoam cups
Microfuge

6. What will you need to do? Prepare double-combed 0.8% agarose gel
Do this several days prior to lab
Can store in plastic ziplock bags with buffer
**Store “wells up” for orientation
Locate dyes
One rack per group
Each group given 1 tube of solutions 1, 2 and 3
Do not throw these away!
Dilute the 20X SB (sodium borate) to 1X SB
300 mL to each group

7. Methods

8. 1. The Digital Micropipette
Micropipettes
Variable small quantities of liquid
Use with proper tip
Firm connection!
Do not lay down or point tip upward with fluid inside
P-20
Dispense liquid only between 2 and 20 μL

9. 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

10. How to use Pipette Aspirating Dispensing Eject tip
Push plunger to first stop
Lower tip below level of solution
Slowly release plunger
Watch solution enter….no air!!
Dispensing
Place pipette tip into tube
Touch tip to side near bottom
Push plunger down to first, then second stop
Do not re-aspirate liquid
Eject tip
Re-use if dispensing same reagent into separate tubes
When in doubt, throw it out!

11. 2. Pipetting – Exercise 1 Locate display window Place tip
Turn knob clockwise to decrease volume
Turn knob counterclockwise to increase volume
Place tip
Use glove!
Do not touch bottom
Practice locating the two stops

12. 3. Pipetting – Exercise 2 Label three reaction tubes (A, B, and C)
Add solutions as directed in table
Fresh pipette tip between each addition
Set pipette to 10 μL, fresh tip, and check volume of each tube
Save for next portion of lab
Tube
dH2O
Soln 1
Soln 2
Soln 3
Total volume A
2 μL
4 μL
10 μL B
8 μL C

13. 4. Gel Electrophoresis
Method uses an electrical current and gel matrix to separate DNA
Molecules are negatively charged
Move towards positive (red) electrode
More negative = move faster
Sort out according to size
Shape
Degree of electro-negativity

14. Using Gel Electrophoresis to Separate Molecules
Agarose gel
Polysaccharide
Bring to gel box
Position so wells located toward negative (black) electrodes
Cover with 1X SB buffer
Sodium borate

15. Using Gel Electrophoresis to Separate Molecules
Set micropipette to 10 μL and load each sample
Record the wells and what is put into each well:
Loading samples:
Center pipette
Depress plunger slowly
Use two hands!

16. Add DNA samples and ladder to the wells and “run to red!”
You RUN TO RED because DNA holds a slightly negative charge, and when electrical current is added to the system, the DNA will migrate to the positive end.
Add DNA samples and ladder to the wells and “run to red!”

17. Using Gel Electrophoresis to Separate Molecules
Close cover
Connect electrical leads
Negative – black
Positive - red
Turn on power and set voltage to v
Check for bubbles
After 3 minutes, check that dyes are moving
Turn off after roughly 10 minutes
Should see all three dyes
Turn off power and record results

18. What will travel where? Orange G – 452 MW BB – 669 Xylene - 538
Solution 1
Solution 2
Solution 3 BB
Xylene OG

19. Conclusions
You should have guessed that Xylene Cyanol was the largest from the results
BUT……. Bromophenol Blue has groups that make it very negatively charged so it is “pulled on” harder by the + electrode

20. Extras

21. 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

22. 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

23. -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

24. 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

25. 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.