An Introduction to Microvolumetrics and Pipetting Laboratory 1

Do Now What is a Chimera? How is biotechnology used? Objectives Thanks Intro Materials Methods Results Closing What is a Chimera? How is biotechnology used? What is a microliter? How do we measure small quantities?

Do Now Which pipette would you use for: 190 µl 25 µl 200 µl Objectives Thanks Intro Materials Methods Results Closing Which pipette would you use for: 190 µl 25 µl 200 µl Determine these window settings: P-1000 P-200 P-20 1 7 5 ? µl

Objectives Purpose: Familiarize students with small volumes Do Now Objectives Thanks Intro Materials Methods Results Closing Purpose: Familiarize students with small volumes Introduce proper use of pipettes Practice loading and running gels

Thank you! Do Now Objectives Thanks Intro Materials Methods Results Closing Any student that Helped to create the buffer solution and “pour” the electrophoresis gels after school!

Introduction Do Now Objectives Thanks Intro Materials Methods Results Closing 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 = 0.001 mL 10 μL = 0.01 mL 100 μL = 0.1 mL 1000 μL = 1 mL

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

Materials Do Now Objectives Thanks Intro Materials Methods Results Closing Kim wipes Disposable bags Spatula Gloves Styrofoam cups Microfuge

What we did to prepare? Do Now Objectives Thanks Intro Materials Methods Results Closing Prepare double-combed 0.8% agarose gel Can store in plastic ziplock bags with buffer 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

Methods

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

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

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!

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

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

4. Gel Electrophoresis Do Now Objectives Thanks Intro Materials Methods Results Closing 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

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

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!

Loading

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!”

Using Gel Electrophoresis to Separate Molecules Close cover Connect electrical leads Negative – black Positive - red Turn on power and set voltage to 130-135 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

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

Closing Purpose: Familiarize students with small volumes Do Now Objectives Thanks Intro Materials Methods Results Closing Purpose: Familiarize students with small volumes Introduce proper use of pipettes Practice loading and running gels

Extras

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

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

-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

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

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.