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An Introduction to Microvolumetrics and Pipetting Laboratory 1.

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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  Solution 1  Solution 2  Solution 3  Distilled water (dH 2 O)  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-20 P-200P 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)

10 How to use Pipette  Aspirating  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  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 TubedH 2 OSoln 1Soln 2Soln 3Total volume A2 μL 4 μL μL 10 μL B2 μL μL μL C2 μL μL 10 μL

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!”

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 Solution 1Solution 2Solution 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 1 Twist dial to desired volume 2 Add disposable pipette tip 3 Press plunger to first stop 4 Insert pipette tip into solution to be transferred transferred 5 Slowly release plunger to retrieve liquid 6 Move pipette tip into desired tube 7 Press plunger past first stop to second stop to transfer liquid, keep the plunger down to transfer liquid, keep the plunger down as you remove it from the tube. 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 dH 2 O.  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  C 1 V 1 = C 2 V 2 (c=concentration, v=volume)  C 1 = desired concentration = 1X  V 1 = desired amount =.5L = 500mL  C 2 = stock solution (what you have) = 20X  V 2 = Unknown (solve for)  Equation: (1X)(500mL) = (20X)(?mL)  ?mL = 25ml  Bring to volume with 475 mL dH 2 O

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.


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