Presentation on theme: "An Introduction to Microvolumetrics and Pipetting"— Presentation transcript:
1An Introduction to Microvolumetrics and Pipetting Laboratory 1
2Overview Purpose: Familiarize students with small volumes Introduce proper use of pipettesPractice loading and running gels
3Introduction Working with extremely small quantities Why necessary? Very high cost of purifying enzymes and DNAHence, need to use instruments to handle small quantitiesVolumes measured in:Microliters (μL)1 μL = mL10 μL = 0.01 mL100 μL = 0.1 mL1000 μL = 1 mL
5Materials Kim wipes Disposable bags Spatula Gloves Styrofoam cups Microfuge
6What will you need to do? Prepare double-combed 0.8% agarose gel Do this several days prior to labCan store in plastic ziplock bags with buffer**Store “wells up” for orientationLocate dyesOne rack per groupEach group given 1 tube of solutions 1, 2 and 3Do not throw these away!Dilute the 20X SB (sodium borate) to 1X SB300 mL to each group
81. The Digital Micropipette MicropipettesVariable small quantities of liquidUse with proper tipFirm connection!Do not lay down or point tip upward with fluid insideP-20Dispense liquid only between 2 and 20 μL
9Reading 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 µL50 µL500 µL
10How to use Pipette Aspirating Dispensing Eject tip Push plunger to first stopLower tip below level of solutionSlowly release plungerWatch solution enter….no air!!DispensingPlace pipette tip into tubeTouch tip to side near bottomPush plunger down to first, then second stopDo not re-aspirate liquidEject tipRe-use if dispensing same reagent into separate tubesWhen in doubt, throw it out!
112. Pipetting – Exercise 1 Locate display window Place tip Turn knob clockwise to decrease volumeTurn knob counterclockwise to increase volumePlace tipUse glove!Do not touch bottomPractice locating the two stops
123. Pipetting – Exercise 2 Label three reaction tubes (A, B, and C) Add solutions as directed in tableFresh pipette tip between each additionSet pipette to 10 μL, fresh tip, and check volume of each tubeSave for next portion of labTubedH2OSoln 1Soln 2Soln 3Total volumeA2 μL4 μL10 μLB8 μLC
134. Gel ElectrophoresisMethod uses an electrical current and gel matrix to separate DNAMolecules are negatively chargedMove towards positive (red) electrodeMore negative = move fasterSort out according to sizeShapeDegree of electro-negativity
14Using Gel Electrophoresis to Separate Molecules Agarose gelPolysaccharideBring to gel boxPosition so wells located toward negative (black) electrodesCover with 1X SB bufferSodium borate
15Using Gel Electrophoresis to Separate Molecules Set micropipette to 10 μL and load each sampleRecord the wells and what is put into each well:Loading samples:Center pipetteDepress plunger slowlyUse two hands!
16Add 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!”
17Using Gel Electrophoresis to Separate Molecules Close coverConnect electrical leadsNegative – blackPositive - redTurn on power and set voltage to vCheck for bubblesAfter 3 minutes, check that dyes are movingTurn off after roughly 10 minutesShould see all three dyesTurn off power and record results
18What will travel where? Orange G – 452 MW BB – 669 Xylene - 538 Solution 1Solution 2Solution 3BBXyleneOG
19ConclusionsYou should have guessed that Xylene Cyanol was the largest from the resultsBUT……. Bromophenol Blue has groups that make it very negatively charged so it is “pulled on” harder by the + electrode
21Micropipette Use Twist dial to desired volume Add disposable pipette tipPress plunger to first stopInsert pipette tip into solution to betransferredSlowly release plunger to retrieve liquidMove pipette tip into desired tubePress plunger past first stop to second stopto transfer liquid, keep the plunger downas you remove it from the tube.8. Eject tip
22Teacher 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 =1XV1= desired amount = .5L = 500mLC2= stock solution (what you have) = 20XV2 = Unknown (solve for)Equation: (1X)(500mL) = (20X)(?mL)?mL = 25mlBring to volume with 475 mL dH2O
24Making a 0.8% Agarose GelEstimate that each gel tray holds 25mL of agarose gel.1mL = 1gramEquation 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
25Making a 0.8% Agarose Gel1)Estimate that each gel tray holds 25mL of agarose gel.6 gels = 180 mL (I round to 200mL).08 x 200mL = 1.6 grams agaroseA 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.