MD100 Medical Biochemistry I Lab Exercise 1 PDF

MD100 Medical Biochemistry I Lab Exercise 1: Introduction to Techniques – Use of Pipetman, Sample Preparation and Dilutions Fall Semester 2024 Andreou M & Kyriakou TC  Objectives ▪ Introduction: Lab characteristics/guidelines ▪ Part A: Use of...

MD100 Medical Biochemistry I Lab Exercise 1: Introduction to Techniques – Use of Pipetman, Sample Preparation and Dilutions Fall Semester 2024 Andreou M & Kyriakou TC  Objectives ▪ Introduction: Lab characteristics/guidelines ▪ Part A: Use of Pipetman Theoretical Background and Exercises PART B: Sample Preparation The main units of concentration and Exercises ▪ PART C: Dilutions Dilution Formula and Exercises COVID-19 Safety Protocol for labs ▪ Be exactly on time to avoid throngs of students at the lab entrance. ▪ Always curry with you the protective gear (lab coat with cuffed sleeves, mask, goggles). ▪ Students without any of the gear will not be allowed in the lab. ▪ Hand hygiene is essential. Wash your hands with soap, then put on some gloves. ▪ Disinfect working surfaces before and after experimentation. ▪ In case a student has symptoms such as fever, couch or breathing difficulties please report immediately.  Absences ▪ Mandatory participation ▪ Physical presence or synchronous participation (only after permission from the department is granted) ▪ Only two justified absences are excused (severe health condition or another emergency) ▪ In every case justified absences must be announced before the beginning of the class. ▪ Lab experiments are not to be repeated!  Be prepared in advance ▪ Study the uploaded material. Print out the protocol or experimental methodology. ▪ Mark materials and equipment needed on the protocol. ▪ Be mentally prepared for technically demanding applications. Closed-toe shoes! Wear lab coat, goggles and gloves! Have your hair tight (pony-tail)! No food and drinks!  Keep a Laboratory notebook Take a note on everything while being explained to you. Keep a record of protocol applications marked with dates. Make calculations of reagent concentrations: e.g.C1.V1=C2.V2 Evaluate results and spot what is needed to be changed. How to move and communicate in the lab Move as if in a bubble Know your surroundings Work as a team Raise your hand to ask No fast movements Work in silence  Contact information Email: [email protected] & [email protected] any question, clarification or any other issue! Office: N30 (2nd floor – Medical Building) EUC account policy In case that a student sends email to a faculty member through his/her personal email account please notify them that in order to read the email it must be send through their EUC email account.  Materials and Equipment ▪ Pipetman (P1000, P200, P10) ▪ Pipet tips ▪ Distilled Water ▪ 1.0 M CuSO4 solution ▪ Cuvettes ▪ Balance machine ▪ Spectrophotometer ▪ Weighing boats ▪ Waste containers  Lab Exercise 1: Introduction to Techniques – Use of Pipetman, Sample Preparation, and Dilutions  Use of Pipetman For volumes less than 1 ml, the most common method for measuring liquid volumes is a device known as a Pipetman. Pipettes are basic tools of a fully-equipped laboratory. The Pipetmen used in this course come in three different types: P1000 (P1000 are useful for volumes from 200 to 1000 μl) P100 (P100 are useful for volumes from 10 to 100 μl) P10 (P10 are useful for volumes from 0.5 to 10 μl) 1L = 1000ml and 1ml = 1000μl Use of Pipetman PART A – Use of Pipetman  PART A - How to check the accuracy of your pipette The most common way to check your pipette accuracy is by weighing water. The density of water at room temperature is 1 g/mL. This means that every microliter (µL) should weigh 0.001 g. In other words, if your pipette is accurate, the amount of water you dispense will equal the amount the water weighs. PART A - Use of 1000μl Pipetman You need to weigh accurately the following volumes of distilled water and complete the table. Table 1. PART A - Use of 200μl Pipetman You need to weigh accurately the following volumes of distilled water and complete the table. Table 2. PART B – Sample Preparation  PART B –Sample Preparation In a liquid solution the liquid that forms the bulk of the solution volume is called the solvent. If the solvent is water, then the solution is called an aqueous solution. The substance dissolved in the solvent is called solute.  PART B –Sample Preparation EXAMPLE 1. What is the % by mass concentration of 100g solution when 5 g of NaOH are dissolved in water? PART C – Dilutions  PART C - Dilutions Many solutions used in biochemistry are prepared by the dilution of a more concentrated stock solution. To make a dilution (or series of dilutions), you need to consider both the desired final concentration and required volume of the diluted material. C1V1 = C2V2 C1 is the concentration of the initial solution (stock solution) V1 is the volume of stock solution needed to make the new solution C2 is the desired final concentration (of new solution) V2 is the desired final volume (of new solution)  PART C - Dilutions Parallel dilutions The dilution formula C1*V1 = C2*V2 can also be applied to solutions that we do not exactly know their concentration, but we know how concentrated they are. For instance, solutions can be concentrated '10X', '20X', '100X' etc. The factor X in a solution indicates that a solution is concentrated and should be diluted before use at 0.5X, 1X, 2X, 5X, etc. Example: We want to prepare 50ml of 1X NaOH from 10X NaOH solution. How many ml of NaOH 10X do we need for this dilution? PART C - Dilutions