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Biochemistry techniques Spring 2011

Biochemistry techniques Spring 2011 - BCHM464 Spring 2011...

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BCHM464, Spring 2011 Biochemistry Techniques 1 Biochemistry Techniques: Pipetting, Precision and the Standard Curve I. Introduction The ability to accurately and reproducibly measure and transfer small volumes of liquids is critical to obtaining useful results when performing biochemistry experimen ts. Part of today’s experiment is designed to teach you to accurately pipette using the pipetmen supplied in your drawer. In addition, you will become familiar with the use of the Shimadzu UV-1800 UV-visible spectrophotometers that will be used. These skills will be used extensively throughout the semester and you should take this opportunity to practice your technique. PART I: The automatic pipetman Automatic pipettors, such as the Gilson Pipetman, are designed to deliver volumes from 2 to 1000 μL with both accuracy and precision. Disposable plastic tips are used to contain the liquid, such that the pipettor itself should never be contaminated by the liquid sample. The plunger at the top moves a piston inside the pipet to displace a precise amount of air, causing that volume to be aspirated or expelled from the tip. The pipettors will enable you to pipet many small volumes reliably and quickly if handled properly. However, the accuracy and convenience associated with the pipetmen comes at significant cost: $285. You should never expose them to caustic or corrosive liquids and should avoid dropping them or banging them on the bench top. The pipetmen used in this course come in three different types (P1000, P200, and P20). You should make sure that you are using the correct pipetman for the volume you need as described in the table below. Do not attempt to set pipetmen for volumes larger than their maximum or for volumes less than zero ; doing so will damage the pipetman. Pipetman Appropriate Volume Appropriate Tip P-20 2 20 μL yellow P-200 20 200 μL yellow P-1000 200 1000 μL blue Proper use of the pipetman will increase your accuracy in pipetting and improve the quality of your results. To become accustomed to the pipetman, you should practice. First, adjust the
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BCHM464, Spring 2011 Biochemistry Techniques 2 volume by turning the volume adjustment knob. Attach a disposable tip to the shaft of the pipetman. Press firmly (but not too hard) with a slight twisting motion to ensure an airtight seal. Now you are ready to pipet. As you depress the pl unger, you will feel a sudden increase in resistance. This is the first “stop.” If you continue pushing, you will find a point where the plunger no longer moves downward, the second “stop.” To use the pipetman, depress the plunger to the first stop, place the tip into the liquid, and allow the plunger to move upwards in a slow, controlled manner . (Do not simply release the plunger as this results in inaccurate volumes and contamination of the pipetman!) Insert the liquid-filled tip into the receiving container, depress the plunger to the second stop to completely expel the liquid. Discard the tip by pressing the tip ejector.
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Biochemistry techniques Spring 2011 - BCHM464 Spring 2011...

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