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Lecture 2

Laboratory Exercise - Week 2.pdf

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University of Toronto Scarborough
Biological Sciences
Daman Bawa

Brunt 2011 Adapted from Riggs B12 manual 2007 BIOB12HSummer 2014Week 2 Basic laboratory techniques continued Use of Pipettes and micropipettes Spectrophotometry quantification of DNA and protein and bacterial media preparationOutline of Lab 2A first lab of the weekMeasuring liquidsSpectrophotometry protein quantificationDNA quantificationOutline of Lab 2B second lab of the weekData analysisclass discussion of spectrophotometry exerciseMaking solutions bacterial media preparation for Week 3 laboratory on bacterial growth Introduction on how to measure liquids In a biology laboratory it is common place to measure liquids The volumes needed can range from liter to ml to microliters Graduated cylinders are used for volumes from approximately 10 ml to a liter quantity Glass pipets are used for volumes from 01 ml to 10 mlFor volumes below one ml one can use micropipettesFor volumes below 01ml a micropipettors is an absolute requirement for accurate measurement The results of your experiment will depend on the art of measuring liquids Specifically the art of pipetting It is a learned skill that takes practice Remember the prefixes milli and micro are used for both liquids and solids based on the gramMilli refers to thousands and micro refers to millionsHere are a few conversions6 liters1000000ul1 liter1ul1031ml 10 liters 1 000ml 1 liter Digitalmechanical micropipettors In research labs amounts of liquid that are 100ul or less are routinely measured by employing micropipettesIt is common place to measure volumes of 1 land even volumes as low as 01l are often requiredThese micropipettes are precision instruments and are very expensive and one of the molecularbiochemistry biologists main toolsThe instrument is a very accurate pump to which a disposable 1Brunt 2011 Adapted from Riggs B12 manual 2007 pipette tip is attachedBy depressing the plunger a volume of air is expelled from the pistonThe tip is then immersed in liquid and the plunger is gently allowed to return to its stop positionThis creates a vacuum in the sleeve and as the piston is released the liquid is pulled up into the tipThe liquid can then be transferred to another tube or solutionThe plunger can be adjusted in order to adjust the amount of air displaced and thus the volume of liquid drawn into the tip Micropipettors can be purchased from many different suppliersIn general to measure liquids from 1ul to 1ml three different adjustable micropipettors are usedThese are called the P10110 lP20 1 although not as accurate as P1020ul the P200 20200ul and the P1000 2001000ul There are also micropipettors that can accurately measure volumes below 1 ul called P101 AND 1LThese are capable of accurately delivering volumes in their working rangesFor example if you are required to dispense 135ul you would use the P20 set to this valueLikewise 54ul requires the use of the P200 Other types of micropipettors can havefixed volume eg 20ul only or are capable of only a few gross changes in the volume dispensed eg an instrument which can be set to deliver 10 20 30 40 or 50ul by having five fixed displacement stops When you need to use a micropipettor be sure of what kind you have and how it worksIf in doubt ask your TAHere are some things to be aware of In the laboratory we have generallyp20 p200 and p1000 type micropipettors Never adjust the micropipettor for use outside of its working range either higher or lowerThis will result in damage to the internal parts Never use a micropipettor without a tip in placeLiquid in the barrel will be drawn into the piston causing loss of seal and perhaps corrosion of the metal partsWith liquid in the tip always keep the micropipettor in an upright positionIf the instrument is placed on the bench or inverted the liquid will run down the tip and into the barrel It is best to store micropipettors in an upright position in a rackNever let the plunger snap backThis causes damage to the internal parts and can also lead to liquid being forcibly drawn into the barrelWhen you put the micropipettor down place it on the bench away from the edge so it wont accidently get knocked off and place itwhere it wont get any spilled liquid on itBe careful not to drop theinstrument We have several different types of micropipettors in the B12 lab Below are some general instructions on how to use them but your TA will give you a demonstration for the type you might be given Make sure you have a demonstration before using 2
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