02 BASIC LABORATORY SKILLS_2024.pdf

BASIC LABORATORY SKILLS Department Of Molecular Medicine School Of Medicine And Dentistry KNUST  Outline 01 02 03 BASIC PIPETTE VS PRINCIPLES LABORATORY PIPETTING SKILLS? 04 05 06 TYPES, P...

BASIC LABORATORY SKILLS Department Of Molecular Medicine School Of Medicine And Dentistry KNUST  Outline 01 02 03 BASIC PIPETTE VS PRINCIPLES LABORATORY PIPETTING SKILLS? 04 05 06 TYPES, PARTS, TYPES OF SOURCES OF AND PIPETTE PIPETTING PIPETTING ACCESSORIES ERROR  Basic laboratory skills Definition: Skills needed to survive in the biochemistry lab. Repeatability and Reproducibility Accuracy and Precision 3 Repeatability and Reproducibility Repeatability: degree of agreement between the results obtained by the same observer under the same conditions with same instruments, at the same location. 4 Reproducibility: degree of agreement between the results of experiments by different individuals, under different conditions, at different locations. 5 Accuracy: How close the result of a measurement, calculation, or specification is to the correct or true value Precision: Refers to the closeness of two or more measurements to each other 6 Low accuracy Low accuracy Low precision High precision High accuracy Low precision High accuracy High precision 7  Most Often Measuring of volumes using pipettes Accuracy and Precision in Pipettes and pipetting 8 Pipette vs Pipetting 9 Principles 10  Glass pipette Types of Pipettes Variable/Micropipette Droppers/ Pasteur pipette 11 Graduated/Glass pipette 12 13  Glass Pipette Fillers Rubber bulb pipette fillers Plastic pipette fillers 14 Rubber Bulb Pipette Fillers 15 Plastic Pipette Fillers 16 Variable (Micro) pipette Single channel Multichannel 17  Pipette Tips > 1000 µL 100 – 1000 µL 10 – 200 µL 0 – 10 µL 18 Parts of Micropipette 19 Forward Pipetting 20 Reverse Pipetting 21 22  SOURCES OF ERROR IN PIPETTING Pipette tips not well fitted to the pipette Plunger pushed beyond the correct (first/second) point of resistance All liquid not dispensed into receiving tube Very fast/slow pipetting 23 VALIDATION OF A PIPETTE 24 PERCENTAGE ACCURACY For 1000µl pipette set to 900µl Five pipetting values = 910µl, 887µl, 882µl, 902µl, and 921µl. Mean = 900.4 µl Accuracy of 99-101% is an indication of good pipetting 25 PERCENTAGE PRECISION For 1000µl pipette set to 900µl Five pipetting values = 910µl, 887µl, 882µl, 902µl, and 921µl. ∑ 𝒙𝒊 −µ 𝟐 standard deviation (SD)= √ ) 𝑵 Xi= each measurement µ= mean of all recorded measurements N= number of recorded measurements standard deviation = 16 Mean = 900.4 µl 𝐒𝐭𝐚𝐧𝐝𝐚𝐫𝐝 𝒅𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 Precision (%) = × 𝟏𝟎𝟎 𝑴𝒆𝒂𝒏 𝒗𝒐𝒍𝒖𝒎𝒆 Higher % means less precise measurements. % precision of 0-2 % is an indication of good pipetting. 26  PROTOCOL Weigh and tar the mass of the beaker Set the pipette to its maximum volume. Aspirate the sample (distilled water). Dispense into the beaker on the balance Record the mass Make a triplicate of the above procedure Convert the three masses achieved to their corresponding volumes in µL 27 Tests Mass of aspirated Mass to Volume sample (µL) (g) 1 2 3 28  POST LAB QUESTIONS With the volumes attained, determine the percentage accuracy and precision of your pipetting (Run the SD to the nearest whole number). Explain your results in terms of the degree of percentage accuracy and precision. State two advantages and disadvantages each of forward and reverse pipetting. 29 THANK YOU 30