Presentation on theme: "Using a Micropipette Huntington Gardens July 2013 MOLECULAR BIOLOGY - THE STRUCTURE OF DNA (INTRO)"— Presentation transcript:
Using a Micropipette Huntington Gardens July 2013 MOLECULAR BIOLOGY - THE STRUCTURE OF DNA (INTRO) http://www.teachersdomain.org/asset/biot11_vid_struct ureofdna/ GENETIC ENGINEERING AND WORKING WITH DNA (INTRO) http://www.teachersdomain.org/asset/biot11_vid_gene ngdna/ MICROPIPETTING (LAB1) http://www.teachersdomain.org/asset/biot11_vid_using micro/
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
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, watch liquid stick to wall to transfer liquid, watch liquid stick to wall of tube. Remove tip, then release plunger. of tube. Remove tip, then release plunger. 8. Eject tip
Reading a Pipette P-20 5.0 µL For Lab 1: Only set out P-20’s Pipette Limits: Never below 2.0 µL Never above 20 µL
Reading a Pipette P-20 P-200P-1000 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)
Two 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.
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
-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
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.
Tips: 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.
Please read through and complete Lab 1 Keep 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
Loading Gels: Insert pipette tip: Under buffer level Above gel well PIPETTING LIQUID INTO THE CENTRIFUGE TUBE (LAB 1) http://www.teachersdomain.org/asset/biot11_vid_smallvolume/ LOADING A GEL (LAB 1) http://www.teachersdomain.org/asset/biot11_vid_agaroserun/
RUNNING A GEL http://www.teachersdomain.org/asset/biot11|http://www.teachersdomai n.org/asset/biot11_vid_isolatedna/
Micropipette tip should be ABOVE the well NOT IN IT!!!!
NICE! Pipette tip is under the buffer level, but above the gel
Lab 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