本期內(nèi)容是 實驗一：實驗設(shè)計?的實驗手冊，實驗?zāi)M請看下一期。本部分內(nèi)容來自 University of California, Berkeley - UC Berkeley Extension, 虛擬實驗的內(nèi)容來自 Labster. 本部分內(nèi)容均不會標記為為原創(chuàng)，但由于是UP主購買的課程，因此不接受非授權(quán)的轉(zhuǎn)載，謝謝您的理解。

每一個生物基礎(chǔ)實驗均會分為三部分：第一部分為實驗的生物理論；第二部分為實驗的指導(dǎo)手冊；第三部分為 Labster?的虛擬實驗?zāi)M。第一部分的基本信息由 Ying Liu, Ph.D. 提供，第二部分的實驗手冊來自 Labster, 第三部分的實驗?zāi)M過程由UP主操作。

Lab 1: Virtual Lab Manual: Experimental Design

Synopsis

In this simulation, you will learn how to design a scientific experiment. As a pharmaceutical detective, you have the chance to perform experiments with human volunteers, animals, and living human cells. Make sure that you choose the correct experimental model to design a scientifically sound experiment for testing the effect of the suspicious drug.

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The scientific method

Humans have always strived to explain natural phenomena. In the first mission of the experimental design simulation, you will learn how to use the scientific method to investigate phenomena, acquire new knowledge, or correct and integrate existing knowledge. Every tool you need to use is available in this virtual lab.

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Design an experiment and test your hypothesis

In your next mission you have the freedom to design your own experiment from scratch while examining biopsies under a microscope. Define your scientific question and choose the right model to test your hypothesis. And don’t be afraid to make mistakes – in this virtual simulation you can repeat the experiment as many times as you want.

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Experimental controls

You will notice that there are many experimental variables in your experiment. Learn how to adjust them and understand why it’s so important to use experimental controls to verify your results. These controls will let you conclude ?whether a suspicious compound is the reason behind the reported epidemic. You’ll get to investigate the effect of different compounds and various concentrations of the same compound.

Will you be able to set up an experiment that can prove your suspicions?

Learning Objectives

At the end of this simulation, you will be able to…

????●?Explain and apply the scientific method

????●?Design an experiment and test a hypothesis

????●?Correctly use experimental controls

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Techniques in Lab

●?Apoptosis assay

Theory

Scientific method

The scientific method is a framework used to accurately describe the world.

Humans have always strived to explain natural phenomena. Historically, explanations of the natural world have been provided by legends and myths. For example, the lack of rain during an El Ni?o year was accredited to angry gods who demanded offerings.

In contrast with dogmatic world views, such as religious explanations, the scientific method is a continuous process. The scientific method consists of techniques for investigating phenomena, acquiring new knowledge, or correcting and integrating previous knowledge.

Hypothesis

The hypothesis is a proposed explanation for a phenomenon based on observations and existing knowledge. It is the initial building block of the scientific method.

The hypothesis leads to predictions, which can be tested through experiments. These three elements can be combined to state the research hypothesis:

If?'hypothesis', and?'method', then?'prediction'.

Predictions are often written as "If X then Y" statements. X and Y stand for the independent and dependent variables, respectively.

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Experiment

An experiment is a procedure designed to test a hypothesis.

Anyone can design a scientific experiment. A proper experiment must be reproducible and reliable, include a control, and use well-designed variables.

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Model organisMs

Model organisMs are species that are used to study certain aspects of biology.

Following is a list of common model organisMs:

? ? Prokaryotes:?●?E. coli

? ? Eukaryotes:?●?Yeast ●?C. Elegans ●?Fruit fly ●?Arabidopsis thaliana ●?Chicken ●?Mouse

Each of these has specific advantages. Depending on your experiment, you can choose which one to use. Check out this list of advantages of model organisMs?and figure out which are important for your experiment!

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Experimental variables

A variable is any factor, trait, or condition that can differ. An experiment?usually has three kinds of variables: independent, dependent, and controlled.

????●?The independent variable is controlled by the scientist.

????●?The dependent variable changes, depending on the independent variable. This is measured by the scientist to prove or disprove their hypothesis.

????●?The controlled variables are conditions which must be kept controlled and constant throughout the experiment so that they don't interfere with the dependent variable. When the experiment is repeated, the controlled variables must be exactly the same.

Cell death assay with fluorescent dyes

Dead cells can be differentiated from living cells by using fluorescent dyes.

When a cell dies, the plasMa membrane disintegrates and allows certain dyes to penetrate the cells. You can use these fluorescent dyes to stain a cell culture and determine the ratio of dead vs. living cells with a fluorescent microscope.

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Fluorescence Microscopy

Fluorescence microscopes?take advantage of the difference in emission and transMission wavelengths of fluorophores?to produce high contrast images. There are several fluorescent dyes?that can freely diffuse into a living cell. Other fluorophores include fluorescent protein tags such as green fluorescent protein (GFP) or SNAP tags that can be expressed transgenically and fluorescently labeled antibodies that bind to molecules of interest in the cell. Therefore, fluorescent microscopy can be used to visualize microscopic processes that are impossible to visualize with other microscopy techniques.

Experimental controls

A control is a comparison.

In an experiment, you test a hypothesis by adjusting something - an independent variable. A control is something you put through the same procedure, without adjusting that independent variable. You can then compare the results of the control to the other results of the experiment.

We will use an Analogy to explain the concept of experimental controls

Imagine you are standing on a hill, which is surrounded by several other similar hills and you start wondering if it would be possible to climb one of those nearby hills with your shoelaces tied together – an obvious thought when one looks at a beautiful panorama!

Luckily, you don’t have to wonder for long because you are accompanied by three boy scouts.

You choose the following experimental setup.

Each boy scout will walk up a hill and light a fire as soon as they reach the top. This way you will be able to see and record the result from a distance.

You are a skilled scientist and know that you have to introduce controls to support your results. Using experimental controls is the way of knowing if your results are due to the variable you are testing, or caused by the experimental procedure itself.

Following the example, you have no idea what awaits your little boy scouts down in the forest. There could be a river or a hungry bear in that valley. You would never know if it were the tied shoelaces that stopped them from making the fire or something else. To avoid such uncertainties, you decide to send off one of the boy scouts as a positive control. You won’t tie his laces together. This should guarantee that he would be able to make it up to one of the hills and light a fire. If there is no fire on that hill, you know there is something wrong with your experimental setup. This is an excellent positive control.

For creating your negative control, you become very creative and find an interdisciplinary approach directly from the Mafioso Repertoire. You fill a bucket with concrete and use it to immobilize one boy scout. You know with such a weight on his feet he might be able to hop down the valley, but he will never make it up the other hill.

Again, you have no idea what will happen down in that valley. You ensured that the shoes of all your boy scouts are well tied and can’t be easily taken off. But in the case they do find a way to take off the shoes and walk up barefoot, you know the boy with his cement shoes would do the same and there would be a fire on his hill as well.

Perfect, this is all you need to run the experiment. With such an excellent setup, you can lean back and wait for the fire signals.

Whatever experiment you are performing, you should always come up with good controls that are truly comparable to the experimental sample. Sending the positive control off with a helicopter would be a useless positive control, just like tying the third boy scout to a tree would be a bad negative control.

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Analgesic Nephropathy

Analgesic nephropathy is an injury to the kidneys caused by Analgesic medications such as aspirin, phenacetin, and paracetamol.

The kidney injuries seem to result from decreased blood flow to the kidney, rapid consumption of antioxidants, and subsequent oxidative damage to the kidney epithelial cells. This kidney damage may lead to

????●?progressive chronic kidney failure

????●?abnormal urinalysis results

????●?high blood pressure

????●?anemia

A sMall proportion of individuals with Analgesic nephropathy may develop end-stage kidney disease.

感謝閱讀！下一期將進行 Labster 實驗！