Presentation on theme: "Using a Micropipette Huntington Gardens July 2013"— Presentation transcript:
1Using 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
2Micropipette 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
3Micropipette Use 1. Twist dial to desired volume . Add disposable pipette tip. Press plunger to first stop. Insert pipette tip into solution to betransferred. Slowly release plunger to retrieve liquid. Move pipette tip into desired tube. Press plunger past first stop to second stopto transfer liquid, watch liquid stick to wallof tube. Remove tip, then release plunger.8. Eject tip
4Reading a PipetteP-20For Lab 1: Only set out P-20’sPipette Limits:Never below 2.0 µLNever above 20 µL5.0 µL
5Reading 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
6Two 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
8Teacher 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 =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
10Making 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
11Making 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.
12Tips: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.
13Lab 1: An Introduction to Microvolumetrics and Pipetting Please read through and complete Lab 1Keep 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
14Loading Gels: Insert pipette tip: Under buffer level Above gel well PIPETTING LIQUID INTO THE CENTRIFUGE TUBE (LAB 1)LOADING A GEL (LAB 1)
20Lab 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