Working in a Biology lab requires a diverse set of skills. Some biologists spend most of their time outside or in the field, while others are in a lab with a microscope or in an office with a computer. Throughout this semester, you will have the opportunity to practice several essential skills for Biological research in and out of the lab. Today, we will start with one: Pipetting.
Research laboratories often require a significant amount of solution making, which requires the ability to appropriately measure and transport chemicals between vessels. You’ll notice a lot of glassware around the lab that we will be using at various points. When such a time arises, we will provide reminders for appropriate use, but pouring from one container to another is somewhat intuitive.
A bit less intuitive, but still quite intuitive, is the use of the micropipette. The micropipette is a special pipette that was designed to measure small amounts of liquids for solutions that need to be very precise. Some researchers need to use micropipettes repeatedly throughout the day for their work, so they were designed ergonomically to conform to the hand and reduce strain. That’s why you drape the curved flange of the micropipette over your index finger and use your thumb to depress the plunger. This is how the pipette is designed to be used. In addition to the flange and the plunger, some other notable parts of the pipette are the volume adjustment dial and the ejector button for the pipette tips that are used to take up liquids.
Before you grab a pipette from the bench, there are a few rules to remember:
We will pause now to allow you to practice using the pipette. Pipetting accuracy is one of the skills you will be assessed on, and will play an important role in successfully completing the lab. We have prepared two activities for you to complete. To help you practice awareness of your own learning, you will also need to assess your level of competence before and after you spend time practicing. Follow the instructions below.
Accuracy by mass of water: One of the best ways to practice pipette accuracy is to choose a volume of pure water and pipette it onto a scale to measure its mass. Water has a very easy conversion factor where 1 mL of pure water (volume) = 1 g pure water (mass). Thus, if you pipette 172 µL (0.172 mL) of water onto a scale, it should weigh 0.172 grams. To get a benchmark for your starting accuracy, record the mass of 10 repetitions of 100 microliters of water, using both the P-100 and P-1000 pipettes, in the tables below.
Mass of 100 microliters of water using the P-100 Pipette
[INSERT TABLE HERE]
Mass of 100 microliters of water using the P-1000 Pipette
[INSERT TABLE HERE]
Now that you have a starting point, you can choose different volumes and different pipettes (the volume should be within the pipette’s range!) to test your accuracy in preparation for your skills assessment. Try to get 5 rounds in a row of a mass within 0.005 g of the intended volume. Use the following space to record your volumes and masses or to take notes as you practice. Record your observations about your strengths and areas for growth with this skill once you feel you’ve practiced enough. For the purposes of scientific inquiry, you should also record the amount of time you spend on this activity. We’ll use it later!
NOTES:
STRENGTHS:
AREAS FOR GROWTH:
TIME SPENT ON THIS ACTIVITY:
For this activity, you will practice the following pipetting rotations using both pure water and water with syrup in the larger and smaller pipettes. You should try to complete all four successfully. These rotations involve adding and taking away liquid in equal total volume but in different increments. Thus, in the end, if you’ve pipetted accurately, your tube should have no traces of water. Viscous liquids require special care when pipetting. Be sure to take your time depressing the plunger! Record your observations about your strengths and areas for growth with this skill once you feel you’ve practiced enough. Record the amount of time you spent on this activity!
P-1000: Add 540 µl to a tube Add 460 µl to the same tube Remove 320 µl from the tube Remove 680 µl from the tube
P-100: Add 23 µl to a tube Add 63 µl to the same tube Remove 50 µl from the tube Remove 36 µl from the tube
STRENGTHS:
AREAS FOR GROWTH:
TIME SPENT ON THIS ACTIVITY:
Final accuracy by mass: Now that you have completed both activities as accurately as you can, it’s time to record some final data on your accuracy. Regardless of your current pipette volume (P- 100 or P- 1000) you will be pipetting 100 µL of water. In the table below, record the mass of the water you’ve pipetted across 10 trials with each pipette (P-100 and P-1000). We will analyze these data as a group.
Mass of 100 microliters of water using the P-100 Pipette
[INSERT TABLE HERE]
Mass of 100 microliters of water using the P-1000 Pipette
[INSERT TABLE HERE]
Your final task in this lab is to find a group of four to reflect and discuss any observations you made about your pipetting skills throughout this process. Record your observations below. We will need them next week.
There is no lab next week due to the labor day holiday. We will return in Week 3 to explore the scientific method and hypothesis formation! During the break next week be sure to read the background information provided on BlackBoard that will explain (1) the project we will be completing throughout the semester, and (2) our expectations as we move forward.
Research across the sciences often relies on a particular order of steps called the scientific method. The scientific method may begin with a question or observation that leads to a hypothesis, or a proposed explanation for the observation, that must be tested through experimentation and analysis to arrive at a conclusion. This method often occurs in a cycle with new questions arising from the completion of every experiment. It is also possible for the steps of this method to be taken somewhat out of order. However as one moves through this process, hypothesis formation and hypothesis testing are two critical parts of doing research in a laboratory.
Hypothesis formation is one step in the scientific method that requires several smaller steps to accomplish. As stated above, a hypothesis is often formed on the basis of an observation the researcher has made of some phenomenon that catches their attention. Below is a guideline for forming a hypothesis based on an observation:
Making an observation that leads to a hypothesis can happen at any time if you’re paying close enough attention! As an example to follow through this guideline, let’s say we decided to plant a tomato garden near the waterfront on campus and a second tomato garden on the other side of campus at the Greens. We plant several healthy tomato vines in the ground in both gardens, but only the garden on the Greens produces fruit. This is an observation that might lead to a hypothesis.
One of the first steps in the process of hypothesis formation is to make sure you have as much information about the phenomenon as possible. If you are planning to conduct a scientific study around your hypothesis, you should always consult primary sources – specifically peer-reviewed scientific literature when possible. This process allows you to understand what we currently know, but also to identify unanswered questions and gaps in our knowledge.
In our example, you might gather information from each of the gardens about the soil conditions and read some scientific literature about how those soil conditions affect tomato plants. You also observe that there are other plants growing nearby without any signs of ill health. There are many possible soil conditions to explore, but science often has to be explored one condition or variable at a time.*
Clearly define the variables involved in your study. In any scientific study, there are many variables involved that can affect the outcome of your experiment. For the purposes of hypothesis formation, we often focus on the independent and dependent variables.
Independent Variable: The factor you manipulate or change in an experiment.
Dependent Variable: The factor you measure or observe to see how it responds to changes in the independent variable.
For our example, if we find a paper saying salt in the soil can negatively affect growth in tomato plants, our independent variable would be the amount of salt in the soil, and my dependent variable would be tomato growth. Some studies will have multiple independent and dependent variables, but you should always be able to identify which variables are independent and which are dependent!
Outside of your variables of interest, there are additional factors that might affect your experiment. We call these control variables. Control variables are factors that must be kept constant during an experiment to ensure that you can isolate the effect of your independent variable on your dependent variable. To extend the above example, if, during our experiment on salty soil, we also gave each of the plants a different amount of water or planted them in different types of soil, we would no longer be able to say that any effect we observed is related to the amount of salt rather than the varying water and soil type. Control variables are an aspect of research that one should be constantly mindful of because they can be easy to miss! In this case, it would mean keeping all of your plants in the same exact environment with the exception of the amount of salt in the soil.
Once you have done your reading and you have identified your variables, it’s time to turn what you’ve learned into a research question. The research question is distinct from the initial observation/question in the scientific method because it is more narrow in scope to focus on a specific phenomenon.
For example, your initial observation would have been, “These tomatoes by the waterfront aren’t growing, but the tomatoes on the Greens are growing.” Your initial question would have been, “Why?” However, your research question would be, “How does salt affect fruit production in tomato plants?”
The final step before we form our hypothesis is to use all of the information you have gathered to propose an explanation for the phenomenon you’re observing. What is the causal pathway? The thing that distinguishes a hypothesis is the inclusion of an explanation for any relationship you might observe between your independent and dependent variables.
For our tomato plants, we may look at the literature and find that salt in the soil can impose drought conditions on tomato plants, which prevents them from taking up enough water to grow fruit. Growing fruit requires a lot of water!
It’s time to construct your hypothesis! There are a few rules about hypotheses and their formatting: Any hypothesis should:
On the last rule, many start their process of hypothesis formation by stating their null hypothesis. The null hypothesis, or the statement of no difference, serves as a sort of benchmark for falsification of the alternative hypothesis, or the hypothesis that states a difference based on supporting evidence.
In this case, the null hypothesis would read something like:
“The addition of salt to the soil will have no effect on drought response mechanisms, and thus, tomato growth.”
The format of your alternative biological hypothesis may vary slightly depending on the relationship between your independent and dependent variable.
For a cause-and-effect relationship: “If [independent variable is manipulated], then [dependent variable] will [outcome] because [explanation].”
For a correlational relationship: “There is a significant relationship between [independent variable] and [dependent variable] because [explanation].”
For a correlational relationship where you know the direction (negative/positive) of your effect: “[change in independent variable], will [effect on dependent variable] because [explanation].”
So our hypothesis could be any of the following three:
“If salt is added to the soil, then tomatoes will not grow on a tomato plant because salt induces drought response mechanisms.”
“There is a significant relationship between the amount of salt in the soil and the growth of tomatoes because salt induces drought response mechanisms.”
“Increasing the amount of salt in the soil will decrease the number of tomatoes produced by tomato plants due to salt-induced drought response mechanisms.”
The hypothesis you choose to proceed with will likely depend on how you’d like to design your experiment or what sort of answer you’re interested in. If you simply want to know if adding some salt to the soil will prevent the plant from growing tomatoes, you would choose the cause-and-effect hypothesis. If you want to know more about how exactly variation in the amount of salt affects tomato growth, thus conducting an experiment with more options than just “salt” and “no salt”, you may decide to use the correlational relationship hypothesis. You would use the correlational relationship hypothesis with direction when you have some supporting literature to predict the direction (negative/positive) of the effect.
It’s time to practice using the data you collected last week! Find a group of four students and complete the hypothesis formation worksheet below. Record your answers in your lab notebook.
Data Entry in Google Sheets: To test your hypothesis, we will use the data collected from our pipette lab. Generally speaking, the more data you have, the better. So we will be using the power of Google Sheets to increase everyone’s sample sizes. This will also give everyone some practice on how to navigate cloud-based data storage and analysis (Google Sheets) and data storage and analysis within a local, desktop application (Microsoft Excel). Generally speaking, these two platforms are extremely similar with a few minor differences. Excel is the more “powerful” of the two, with more graphing capabilities, data analysis extension packages, and other add-in features. However, Google Sheets allows for collaborative data entry and analysis. For the purposes of this lab and most of your future labs, you will need to be adept at moving between these two platforms. Take 5 minutes to discuss which data you would like to be entered into our lab spreadsheet, and we will practice this process together!
Be sure you are logged in with your SMCM ID and password!
Data Analysis in Excel: One of the first steps in quantitative (data with numbers) data analysis is to take a look at your “summary statistics”. The summary statistics are a collection of calculated values that summarize your data. You will find a description of the relevant summary statistics for this lab below. Be familiar with each one as it will be on your skills assessment!
Manually calculating summary statistics in Microsoft Excel involves using built-in functions to compute measures such as mean, median, mode, variance, and standard deviation. Here’s a step-by-step guide on how to do this:
Step 1: Organize Your Data
Step 2: Calculate the Mean
=AVERAGE(A2:A11) and
press Enter.What it tells you: The mean (average) provides a central value of your data set by summing all the values and dividing by the number of data points.
Step 3: Calculate the Median
=MEDIAN(A2:A11) and
press Enter.What it tells you: The median is the middle value of your data set when it is ordered. It is useful for understanding the central tendency, especially when your data has outliers.
Step 4: Calculate the Mode
=MODE.SNGL(A2:A11) and
press Enter.What it tells you: The mode is the most frequently occurring value in your data set. It is useful for identifying common values.
Step 5: Calculate the Range
=MAX(A2:A11)-MIN(A2:A11) and press Enter.What it tells you: The range provides the difference between the highest and lowest values in your data set, giving an idea of the data’s spread.
Step 6: Calculate the Variance
=VAR.S(A2:A11) for a
sample variance or =VAR.P(A2:A11) for a population
variance, and press Enter.What it tells you: Variance measures the dispersion of your data points around the mean. A higher variance indicates that data points are more spread out.
Step 7: Calculate the Standard Deviation
=STDEV.S(A2:A11) for a
sample standard deviation or =STDEV.P(A2:A11) for a
population standard deviation, and press Enter.What it tells you: Standard deviation quantifies the amount of variation or dispersion in your data set. A lower standard deviation indicates that the data points are closer to the mean.
Step 8: Calculate the Minimum and Maximum
=MIN(A2:A11) and press
Enter.=MAX(A2:A11) and press
Enter.What it tells you: The minimum and maximum values indicate the smallest and largest values in your data set, respectively, helping to understand the full range of your data.
Step 9: Calculate the Sample Size
=COUNT(A2:A11) and
press Enter.What it tells you: The sample size indicates the number of data points in your dataset, which is essential for many statistical analyses.
Step 10: Calculate the Standard Error
=STDEV.S(A2:A11)/SQRT(COUNT(A2:A11)) and press Enter.What it tells you: The standard error measures the accuracy with which a sample represents a population. It is the standard deviation of the sample mean distribution.
Cell References: Make sure your cell references are correct and adjust them if your data range changes.
Consistency: Ensure your data set does not contain any text or blank cells that could affect the calculations.
By following these steps, you can manually calculate various summary statistics in Excel to analyze and summarize your data effectively. Note that these formulas should apply for data analysis within Google Sheets as well! If you encounter any errors, simply use Google to find the correct formula.
Data Visualization in Excel: Microsoft Excel is more powerful as a graphing tool than Google Sheets, so we will do all of our graphing for this semester in Excel. The final step for today’s lab is to find the best way to display your data in a graph and create that graph using Excel. We are going to provide a bit less guidance on this part because each person’s graph may be different. However, it’s a good rule to make sure your numerical data are displayed in a way that highlights your result and that you include some visual representation of error (eg, error bars; confidence intervals, etc.). Use the graphics flow diagram provided to choose the graph that is best for your data. A completed graph and summary statistics will be your exit ticket for today’s lab. Your TA or instructor will examine your graph and may give you feedback or things to correct. Spend the time making sure you do it correctly the first time! Paste an image of your final graph and summary statistics in your lab notebook.
These are examples from published text. If you reference them, make sure that you cite them accordingly. However, you should not be copying this text, merely using it as a guide. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. All of your writing also needs to be in your own words to avoid plagiarism. You can use this as a guide, while you learn how to write a lab report. Your reports will be run through a plagiarism checker.
For this week, use your hypothesis to write out the “purpose statement” of your introduction. This is generally the last paragraph of your introduction. Use this text as a guide for how to describe the project you will be doing this semester, and why it is important. At the end of the paragraph, be sure to add your hypothesis with proper phrasing and formatting.
“In the present study, we conducted an in-depth analysis of the gene expression of IGF1, IGF2, and the five functional IGFBP genes across seven life stages ranging from preoviposition to adulthood, and six tissue types in the brown anole lizard. To our knowledge, this is the first comprehensive study of IIS network expression across time and tissues in a reptilian model species. We contrast our gene expression patterns in the brown anole lizard to what is documented in other species, with a focus on the laboratory mouse (the most commonly used model species in IIS research) and humans. The data collected in this study are the foundation for further developing the anole as a model species in biomedicine and physiological genomics.”
This week, we will be learning another essential skill for any Biology student, Microscopy. For the purposes of this lab, we will be using microscopes to begin the process of identifying our unknown tissue samples. In all vertebrates, tissues of a similar type have similar structures across species. Thus, the muscle tissue of a human looks very similar to the muscle tissue of a lizard under magnification. There is a whole field of Biology called Histology that is dedicated to the study of different tissues using microscopy. Today, we will be learning how to use a light microscope to examine tissues, measure structures under a microscope using the ocular micrometer, and use histology to form a hypothesis about our unknown tissue types.
Find a slide to practice with. You can choose any of the slides provided.
Now it’s time to use your microscope skills to make some observations that you will use to form a hypothesis about what tissue type your unknown might be. During your preparation for this lab, you will have watched a video about some of the basic structures you might find when looking at different types of tissue under a microscope. You’re going to examine example slides for each of the different possible tissue types (brain, gonad, skeletal muscle, heart, liver), and compare it with a prepared slide of our unknown.
You will spend time sketching and taking photos of each tissue type (skeletal muscle, heart, liver, brain, and gonad) for your lab notebook. Be sure to take notes as you go. Examine the slides at different magnifications, but be careful going up to the 40x! It can break the slides. When recording measurements, sketches, or photos, be sure to note the magnification for context. Use your ocular micrometer to measure any features you think might be useful for differentiating tissues that look similar to one another. The internet can be a very helpful resource here, so if you’re not sure what you’re looking at, google it! You will examine each of the possible tissue types and compare them to your unknown.
During the last lab, we spent some time working on hypothesis formation. We will be adding to your hypothesis formation skills today by writing a hypothesis for identifying your unknown tissue type. The structure of this hypothesis will be very similar to the examples from last week, but your method for testing your hypothesis will be less quantitative (think numbers) and more qualitative (what kind of DNA do we see on the gel). Thus, your dependent will be “The results of sanger sequencing” rather than something like “Number of tomatoes produced”.
Work together with your group to write a hypothesis about the identity of your unknown tissue being sure to include an independent variable, dependent variable, and biological explanation. Record it in your lab notebook and discuss it with your instructor. You will briefly present your hypothesis to the class before you leave.
For this week, start writing your methods and results section of your manuscript. Be sure to describe (1) how you visualized your unknowns in the methods, and (2) what you saw in the results. In the results section, be sure to expand slightly on how your results in the histology section either support or falsify your hypothesis from week 2.
RNA, or ribonucleic acid, is a vital molecule in cellular biology that plays a crucial role in gene expression. It acts as the intermediary between DNA, which contains the genetic blueprint, and proteins, which carry out cellular functions. RNA is transcribed from DNA in a process called transcription, resulting in messenger RNA (mRNA) that carries the code needed to synthesize proteins. By measuring the levels of mRNA, scientists can determine which genes are active and being expressed at any given time. This is essential for understanding cellular responses to various conditions, developmental processes, and disease states, making RNA a key focus in studying gene regulation and function. Our class is particularly interested in RNA and gene expression because some genes are uniquely expressed within certain tissues. So if you have hypothesized that you have a specific tissue type in the class, we can take a gene that we know is highly expressed in that tissue and test for its presence by extracting and analyzing the mRNA present. If you find the gene is being expressed in the tissue, it supports your hypothesis. If you find the gene is not presently expressed in the tissue, it may indicate that your initial hypothesis is on its way to being falsified.
So, how do we isolate nucleic acids from tissue samples? How do we go from having a chunk of liver, or brain, to have DNA or RNA in a tube? Thanks to recently developed methods, it’s probably a bit easier than you think. It also relies heavily on the biochemistry you have gone over in lecture.
In this lesson, you will learn about spin column nucleic acid purification, a technique for isolating DNA or RNA from biological samples. The process begins with lysing the sample to release nucleic acids using something called “lysate”. The lysate is passed through a spin column containing a silica membrane that binds the nucleic acids. After binding, the column is washed to remove impurities. Finally, the purified nucleic acids are “eluted”, or released and collected, from the column. This method is appreciated for its simplicity, speed, and high yield of pure nucleic acids, making it ideal for various molecular biology applications.
We will be using a different brand of RNA extraction kit. However, the protocol is very similar. There are a number of different issues that can arise when doing RNA extractions, all of which will affect your outcome. Some of these are within your control, while others are not. Some of the common issues that you can prevent include:
After we isolate your RNA, we will quantify iit to see how much RNA you were able to get from your sample. In order to do this, we will use the Nanodrop spectrophotometer (‘Nanodrop’). The Nanodrop is a quick way to estimate the quantity and quality of your RNA.
Pure RNA will have a A260/280 ratio of ~2.0. However, DNA has a ratio of ~1.8. Pay close attention to your values, as it can give you a lot of information about what is in your sample. If your value is closer to 1.8 than 2.0, it may indicate DNA contamination. Additionally, a pure sample should have a A260/230 ratio of at least 2.0. Anything below 1.8 indicates significant contamination by other molecules.
The initial step of molecularly verifying your tissue type is to extract the RNA from your sample. The first step toward doing that is to homogenize the tissue, which is done by taking a small piece of tissue, combining it with RLT buffer and a metal bead, and shaking it at a high speed until the sample resembles a liquid consistency. We have done this step for you. The sample you receive will be frozen in this liquid form. You will continue the protocol from this stage.
Remember, these are examples from published text. If you reference them, make sure that you cite them accordingly. However, you should not be copying this text, merely using it as a guide. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. All of your writing also needs to be in your own words to avoid plagiarism. You can use this as a guide, while you learn how to write a lab report. Your reports will be run through a plagiarism checker.
“Samples were lysed in RNAspin Lysis buffer (GE, Cat. No. 25-0500-70) with 5 mm stainless steel beads (Qiagen, Cat. No. 69989) using the Tissuelyser II (Qiagen) at 30 Hz for 3 min. From the juvenile livers, total RNA was extracted with the RNeasy Plus Micro Extraction Kit (Qiagen, Cat. No. 74034). RNA concentration and purity was assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific).”
“Following column-based RNA extraction, the concentration of RNA across the samples ranged from 50 to 200 ng/µL, with an average yield of 125 ng/µL. The purity of the RNA was evaluated by the A260/A280 and A260/A230 ratios, which are indicators of protein and organic compound contamination, respectively. The A260/A280 ratios for all samples were between 2.0 and 2.1, indicating high purity and minimal protein contamination. The A260/A230 ratios ranged from 2.0 to 2.3, suggesting the absence of contaminants such as phenol or guanidine.”
Note: If your values are outside the guidelines for RNA samples, reference the Nanodrop manual (available in BlackBoard) to estimate what type of contamination you may have in your sample and report that in your results.
So far, you have looked at your samples under a microscope and were able to form a hypothesis on what type of tissue you may have as your unknown sample. Last week, we began molecular verification of the tissue type by isolation and extracting RNA from your tissue.
Last week we discussed how different tissues “express” different genes from your genomes in different quantities. What this means, is that we can identify genes that are specific to certain tissue types, or are much more highly expressed in certain tissue types. You will be learning about this process extensively in genetics!
As you know, when possible, we use multiple methods to verify our results. This week, we are moving forward using Polymerase Chain Reaction (PCR) to determine whether or not molecular techniques support or falsify your existing hypothesis. At its core, this process allows us to take a single copy of a section of DNA we are interested in, and make millions of copies in a test tube.
For the purpose of verifying your tissue types, we know that each possible tissue type has a gene that is expressed uniquely to that tissue. We have identified genes that are specific to the possible tissue types used in this lab, and have designed primers that would amplify those genes in your samples if they are present. The genes that are highly related to specific tissues are listed below. However, this process is especially important for you today, as it will allow you to further validate your tissue type. For example, if you hypothesize that you have kidney tissue, and one gene that is highly expressed in kidney tissue is the PKD1 gene, you would expect PCR amplification to lead to many, many copies of PKD1 being produced. If that does not happen, or if you see a gene specific to liver tissue being amplified, your original hypothesis of tissue type may be falsified.
Before lab, be sure to watch the inscriptional videos describing the processes we are using today and complete this table:
Based on your hypothesized tissue type, which gene do you expect to amplify using polymerase chain reaction on your samples, and why?
Now one thing that you may have noticed at this point is that all of your instructional videos mention the need for a “DNA” template in PCR reactions, and that is true! But…. you have RNA, not DNA. So how do we go from RNA to DNA in order to successfully complete PCR?
We have a process available to us called “reverse transcription,” where we can actually reverse the first part of the central dogma, allowing us to make a complementary DNA (cDNA) copy of the RNA you have already extracted last week.
This week, you will complete two very important steps:
The total amount of RNA that goes into a cDNA reaction can vary. The protocol we are using recommends 1ng to 1µg (or 1000ng) of total RNA. We are going to aim to use 500ng of total RNA in our reaction. Now this is going to require a little bit of math. Make sure to record your calculation on your lab notebook page in the calculations section.
You can use the concentration of RNA, which you determined last week on the Nanodrop, to calculate how many microliters of RNA to add to the cDNA reaction. Here is an example:
We want a total of 500ng of RNA in our reaction. The nanodrop indicates we have a concentration of 100ng/uL. This means that in every microliter of volume, we have an average of 100ng of RNA. Therefore, we would need 5µL of RNA in our reaction (500ng ÷ 100ng/µL = 5µL). The formula for this calculation is: Total amount of RNA wanted in the reaction ÷ concentration of RNA sample = required volume in microliters. Calculate the total volume of RNA needed to reach 500ng using your sample concentration. After performing this calculation in your lab notebook, record your result here as well:
Volume needed __________________________
If this results in a total volume of less than 16µL, then you are good to go! If this results in a volume of greater than 16µL or less than 1µL, we need to make a couple of adjustments. Volumes greater than 16µL will not fit in the total reaction volume, and volumes less than 1µL are not reliably measured. To be as precise as possible, use this flow chart to ensure you follow best practices:
Once you determine that you have properly calculated your RNA volumes, you are ready to proceed to setting up the reaction.
Note: If you need to make a dilution of your RNA based on your calculations above, do so now. After your dilution is produced, use this as the template for your cDNA synthesis. If no dilution was required, use your original sample in the volume as calculated.
Note: As you learned during your pre-lab instructional video, PCR requires the use of enzymes to replicated strands of DNA. This means that the reaction is temperature sensitive. You want to set up your reactions, and store your components on ice at all times.
Remember, these are examples from published text. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. This is to be used as a guide, and should not be copied verbatim from the text. Your work will be run through a plagiarism checker upon submission.
“Sample concentrations were standardized by the use of 100 ng of total RNA for cDNA synthesis. Following manufacturer protocols, total RNA (100 ng) was used in cDNA synthesis reactions to create single stranded DNA using qScript XLT cDNA SuperMix (QuantaBio, Cat. No. 95161-500).”
“We surveyed for the presence of gene expression of five IGFBPs (IGFBP1 through IGFBP5) in embryos, juvenile livers, and six adult tissues (Table 2). Primer pairs for IGFBP1 through IGFBP5 were designed by using the green anole (Anolis carolinensis) reference genome (1), using the predicted transcripts from the National Center for Biotechnology Information. Primer pairs were designed to produce a PCR product between 100 and 250 bp in length and were located within an exon so we could verify their ability to amplify DNA if the cDNA reactions did not amplify, thus indicating no expression. Water replaced cDNA as the no template controls. All samples were amplified in 25 µL reactions with final concentrations of 1X IBI Taq Mastermix (IBI Scientific, Cat. No. IB43101), 0.15 µM of each forward and reverse primers, and 1 µL of cDNA at a 1:2 dilution.”
The results section will be written following next week’s lab.
There are no labs during week 7 as we accommodate the college reading days. While there are no scheduled labs, we will be offering open lab hours for you to come and complete skills assessments. Please use this time to catch up if needed.
Now that you have completed your PCR, you have (in theory) taken your gene of interest and replicated it many times. However, the only way to know if your reaction was successful and determine which genes were present in your sample is to visualize the DNA somehow. Right now, any DNA you have is floating in your tube suspended in liquid. Unfortunately, the human eye can not visualize DNA without some assistance. Luckily, we have a molecular tool called gel electrophoresis!
Gel electrophoresis is a fundamental technique in molecular biology used to separate and analyze macromolecules, such as DNA, RNA, and proteins, based on their size and charge. This process involves placing a solution containing the molecules into a gel matrix and applying an electric current. The gel, made of agarose, acts as a sieve, allowing smaller molecules to move more quickly through its pores than larger ones. The electric current drives the negatively charged molecules (DNA) towards the positive electrode. As the molecules migrate through the gel at different rates, they form distinct “bands”, which can be visualized using various staining methods. This separation allows researchers to identify and quantify the molecules, making gel electrophoresis an essential tool for genetic analysis, molecular cloning, and forensic investigations.
You will start today’s lab by producing an agarose gel that you will use to separate out the DNA bands present in your sample.
Once the gel is ready, you have the tricky task of loading the gel. So far you have your PCR product, which may or may not contain your DNA products of interest, in a tube. You also have an agarose gel which contains a stain that binds to DNA, allowing us to visualize it under UV light. Now you need to load your samples onto the gel and let the electrical current flow through the agarose to separate your DNA by size.
Once you run the gel, you will be able to analyze your results. This is going to include looking at your samples under UV light, comparing your samples to the DNA ladder to determine their size, and determining which genes are and are not present in your sample.
Now that we have covered the basics, I think you’re ready to give it a try! Carefully follow these directions to analyze your PCR results.
Remember, these are examples from published text. If you reference them, make sure that you cite them accordingly. However, you should not be copying this text, merely using it as a guide. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. All of your writing also needs to be in your own words to avoid plagiarism. You can use this as a guide, while you learn how to write a lab report. Your reports will be run through a plagiarism checker.
“Amplifications (presence/absence) were verified on 2.5% agarose gels with 1 µg/mL GelGreen (Biotium, Cat. No. 41004). PCR product (10 µL) was mixed with 2 µL of 6X Loading Dye (NEB, Cat. No. B7024S) and run at 120 V for 1 hour.”
“Samples were classified as positive for expression if a band was visible in blue light. If no band was visible, it was considered negative (no expression).”
“All five IGFBPs were expressed at each embryonic stage (Fig. 2, A and B; Supplemental Fig. S3: https://doi.org/10.6084/m9.figshare.12349985). In adults, the expression of each binding protein was detected in every tissue in at least one individual and, in many cases, all individuals (Fig. 2C, Supplemental Fig. S3).”
“Images of the electrophoresis agarose gels used for qualitative categorization of expression can be found in Figure 2.”
While you are required to attend lab this week, we are using the time a bit differently. During this time period, you will be able to practice any skills necessary, take skills assessments, and work on your second round of peer reviews. All students are required to attend lab this week.
Due to advising days, there are no scheduled labs during week 10. The lab will be open for you to come in and complete skills assessments if needed. The schedule for open labs will be made available on BlackBoard.
Sequencing PCR products is a critical step in molecular biology to verify the accuracy and integrity of the amplified DNA. This verification ensures that the amplified product matches the intended target sequence precisely, confirming its accuracy and specificity. PCR can sometimes amplify non-target sequences, particularly if the primers are not highly specific, so sequencing helps ensure that the correct DNA sequence has been amplified and that no non-specific products are present. Additionally, in experiments where mutations or variations are being introduced or studied, sequencing allows for the identification and confirmation of these changes within the PCR product. This step is essential for quality control, ensuring the consistency of the PCR product for downstream applications, such as cloning, gene expression analysis, or genetic engineering. By comparing the sequenced PCR products against reference sequences, researchers can validate that the experimental conditions and reagents used are functioning correctly, thereby enabling precise and reproducible scientific results. In other words, you have a DNA band according to your gel electrophoresis, and you have a good idea of the gene you amplified based on the primers you used and the size of the product, but how are we certain that you amplified exactly what you were expecting?
Today, we are going to prepare our PCR amplifications for genomic sequencing for this very reason – to verify that you have amplified your expected gene successfully. In order to send our samples off for sequencing, they have to be diluted to a very specific range of concentration. You may remember using the Nanodrop a few weeks ago to quantify your RNA extractions. You will be using the Nanodrop again in order to quantify your PCR product and dilute it to a proper concentration for sequencing.
We are sequencing through Eurofin Genomics, who will perform Sanger Sequencing on our samples to ensure that we amplified the gene that we think we amplified. You will be surprised how much it reminds you of PCR!
We have PCR products, which are DNA based. You will be sending off the PCR product that produced a strong band on your gel electrophoresis. There is no need to sequence samples that did not amplify, as there will not be sufficient amounts of DNA to sequence.
You will need to reference the primer table provided to you in Week 5 to determine the size of your amplicon (PCR band size in base pairs). All of our potential primer pairs produce a product between 100 and 300 base pairs (bp), or 0.1-0.3 kilo-bp. Use the table below to determine the concentration of your template DNA for sample submission. If you read the table correctly, you will see that you need to submit a sample between 10 and 20 ng/µL. PCR products are typically far more concentrated than this. Therefore, you will need to determine the concentration of your PCR product using the Nanodrop.
Now that you have found the concentration of your sample, you will need to dilute it to the required concentration of 10-20 ng/µL. We will be aiming for a concentration of 20ng/uL. The formula for calculating a dilution is C1V1=C2V2. You know what your current concentration is, and you know what concentration you are aiming to produce (20 ng/µL) in a total volume of 50 µL. Therefore, you can calculate V1 in the equation to determine the volume of your undiluted sample to add to a fresh 2mL tube. You will then add water to a total volume of 50 µL.
Here is an example for your benefit:
My concentration as determined by nanodrop is 256ng/µL.
C1V1=C2V2 (250ng/µL)(V2)=(20ng/µL)(50µL) (250ng/µL)(V2)=1000ng V2=1000/250 V2= 4µL → This is the volume of your PCR product to put in a fresh tube.
Total volume - PCR product = volume of water 50 µL - 4 µL = 46 µL → You will then add 46 µL of water to the tube and mix it well.
You are now going to prepare your tubes for submission. You will need to submit two separate tubes for each sample. One tube will contain the diluted PCR product pre-mixed with the forward primer, and the second will contain diluted PCR product pre-mixed with the reverse primer.
Remember, these are examples from published text. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. This is to be used as a guide, and should not be copied verbatim from the text. Your work will be run through a plagiarism checker upon submission.
“PCR product from one sample for each gene was sequenced to verify amplification of the target gene. PCR products were diluted to 20ng/µL and sent to Eurofin Genomics for sequencing in the forward and reverse directions using gene-specific primers.”
After we sent our samples to Eurofin for Sanger Sequencing, they ran the samples on their sequencer. As a result, they have returned a number of files associated with your samples. You are going to receive two specific file types that you will analyze: (1) AB1 files, and (2) Histogram depictions of the sequences. AB1 files are binary files generated by automated DNA sequencers. They contain raw data from Sanger sequencing, including:
Histogram files typically accompany AB1 files and represent the distribution of quality scores across the sequenced bases. They provide a visual summary of the sequencing quality. Histograms of quality scores help to quickly assess the overall quality of the sequencing run. A high proportion of high-quality scores (e.g., Phred scores above 20) indicates a reliable sequencing result. You will be looking at your returned sequence files in a program called UGENE.
Within UGENE you will be determining which sequence regions are high enough quality for analysis. Then, once you have looked at your sequences and evaluated their quality, you will run what we call a “BLAST” in UGENE as well. BLAST (Basic Local Alignment Search Tool) is a powerful algorithm for comparing a query sequence against a database of sequences. When Sanger sequencing reads are obtained, researchers often use BLAST to identify the sequence by finding the closest matches in the database. This process helps in:
Remember, these are examples from published text. Make sure you change the values and all relevant product information to be accurate for what you actually did in lab. This is to be used as a guide, and should not be copied verbatim from the text. Your work will be run through a plagiarism checker upon submission.
“The resulting PCR product sequences were verified by BLAST analysis using the NCBI database in Geneious (version 11.1.4) and verified they were the correct targets.”
Be sure to expand on this briefly to describe what this means. Did they match? Did they not match? Does this support or falsify your hypothesis from week 2?
At this stage, you have all of your data! You should now focus on synthesizing all of your findings to write a discussion and conclusions section. Your final peer review is coming up next week, and it will be your only opportunity to receive feedback on your discussion and conclusion.
There are no labs next week as it is Thanksgiving break. The week after, we will be giving presentations that summarize your findings for the semester. Make sure to check the requirements on BlackBoard and come prepared to give your presentations on week 15!