Introduction

Here you will find help on procedures like pipetting and centrifugation as well as simple calculations that are needed throughout the course. These principles and calculations (e.g. how much buffer or water goes into the tube; converting ng to μg and vice versa) are essential to understand, so we will be asking you to demonstrate your understanding of these throughout the semester as part of your lab worksheets and pre-lab quizzes.

We also have a short review section on gene structure and function. This is essential to understand for the course.

A. Units – important to get them right:

It is important to understand the units you are working with and to be able to convert between them quickly. In most cases this involves moving the decimal point so it should come automatically with some practice.

A-1. Units of Weight

These can be measured with a scale in theory but we use other methods like spectrophotometry when dealing with tiny amounts of nucleic acids. Units of weight are measured in grams or fractions of grams. Make note of the prefixes below:

• 1g (gram) = 1000 mg (milligrams, 10^-3 g)
• I mg = 1000 μg (micrograms, 10^-6 g)
• 1 μg = 1000 ng (nanograms, 10^-9 g)
• 1 ng = 1000 pg (picograms, 10^-12 g)
• 1 pg = 1000 fg (femtograms, 10^-15 g)

We will usually not go below ng in our calculations, but it is useful to know the other prefixes. We sometimes use quite tiny amounts in molecular work – and you may well come across picograms or femtograms in other courses/work situations.

A-2. Units of volume

Our volume measures will be liquid measures, and are calculated in mL (millilitres, 10^-3 L). The prefixes are all the same as above, for example:

• 1 mL = 1000 μL (microliter, 10^-6 L)
• 1 μL= 1000 nL (nanoliter, 10^-9 L)

In general we won’t deal with volumes below 1 μl. These cannot be pipetted accurately and it is often better to dilute a concentrated sample to a lower concentration (see concentration, below) and pipette a larger volume, in order to be accurate.

A-3. Molarity

Molarity is calculated the same way for molecular work as for any biochemistry- you use the molecular weight of the molecule to make a solution of the desired molarity. The following video shows an example of making a small volume of 1M NaCl solution. It then talks about dilution which is useful to understand as well.

You will need to log onto LabXchange and create a free account. Once you have successfully logged on, click on the video link below.

https://www.labxchange.org/library/items/lb:LabXchange:758f029f:video:1

A-4. Concentrations

In our work, the concentrations we use vary depending on the materials we are working with. The concentrations we use for enzymes are expressed as units, U per μl; this may be a different amount for each different enzyme (either weight or moles) because the U measure is based on activity. You will learn more about this later when we discuss restriction enzymes. In that case the standard definition of a unit is the amount of enzyme needed to digest 1 μg of DNA in one hour at a specific temperature.

Solutions we use in working with nucleic acids, such as salt, for instance, are usually expressed as molarity, moles per L, as discussed above. Our lab technicians generally make such solutions in advance for the in-person lab, and so we will not require you to make the calculations for such solutions.

In the case of nucleic acids, molarity is not that useful a measure; we use weights instead, most often in ng/μl or μg/μl.

We are going to be emphasizing your ability to work with these concentrations so there will be calculation questions to do most weeks. Most important is being able to figure out from a CONCENTRATION, what VOLUME of a substance, usually DNA or RNA, to add to a reaction. This is something you will have to do in setting up many reactions in the lab.

Most questions you will be asked will provide a “stem” – this will have all the information needed to answer several potential questions that you could be asked.

E.g.: You have an RNA prep that is 45 ng/μl in a total volume of 150 μl. (THIS IS THE STEM from which several questions could be asked, examples below):

i) How much RNA do you have total in the tube?

• For this question you use all the information. If every μl has 45 ng and you have 150 μl of the prep then you have 45ng/μl x 150 μl. The μl cancel and the number of ng is 45 x 150 = 6750 ng of RNA, which is better expressed as 6.75 μg of RNA.

ii) If you want to reverse transcribe the RNA and need 400 ng for the reaction, how much of your prep do you use?

• For this question the concentration of the RNA matters but the amount you have total is not needed. “How much of your prep” is specifically asking how many μl you need to add. So if you have 45 ng in every μl and you need 400 ng total, you divide 400ng/45 ng/μl and the ng cancel, leaving you with 400/45 μl needed. You should be familiar enough with numbers to see that this will be around 9 μl (it comes out to 8.9μl ) That familiarity helps you detect calculation errors. Sometimes you hit the wrong button of the calculator and get an answer that doesn’t make sense, for instance, 89μl . If you realize the number seems off, then you can recheck the calculation and make sure you have it right. Accomplished problem solvers check their answers. The way you can check this answer is to multiply 8.9 x 45 and see that the answer is 400, or very close to 400 because of rounding.

iii) If you need to dilute your RNA prep to 15 ng/μl, how do you proceed?

• For this question, the needed information is the current concentration. If you want to dilute to 15 ng/μl you are decreasing the concentration 3-fold (45/15 = 3). This is a 1 in 3 dilution. So you will add 1 part of your RNA sample, 5 μl for example, and 2 parts ddH₂O , or 10 μl (NOTE: this is commonly done incorrectly. The term 1 in 3 means one part of your substance being diluted into 3 parts TOTAL, so for 1 in 3 dilution it is 1 part RNA plus 2 parts water for a total of 3 parts. A 1 in 5 dilution is 1 part RNA (or whatever the reagent is) to 4 parts water for a total of 5 parts)

A general note for doing a dilution is that it is more accurate to pipette, say 5 μl (rather than 0.5 or 1 μl) of your sample plus whatever amount of water is needed (10 μl in the example above), and to mix thoroughly after diluting, to ensure that the nucleic acid is evenly distributed throughout the sample.

B. Using the correct pipette and basics of how to pipette:

Please watch this short video explaining how to use the micropipettes. It was made by some former TAs and contains the most key information you need.

Please notice that when the video shows how to mix a solution by drawing the solution in and expelling it again an important mistake was made that is useful for you to see. The liquid was drawn into the pipette tip too quickly causing the liquid to splash up onto the micropipette and thus contaminating it. You can see that the blue liquid is on the actual pipette and not just in the tip. If that happens the pipette needs to be carefully cleaned before it is used, to avoid contaminating the solutions you are working with. Let us know right away and we can clean the pipette before any damage is done.

C. Centrifuge use: what is g and how is it related to rpm?

Please watch this video which shows how to use a centrifuge quite similar to what we will use in the lab in person. It also explains what g is (relative centrifugal force, rcf) and how it relates to rpm (rotations per minute). The rpm you use to achieve a particular rcf will depend on the diameter of the rotor you use and so will differ from one type of centrifuge to another. That is why protocols usually give the centrifugation in g; it allows you to calculate the rpm for whatever centrifuge you have.  We have a posted list of rpm and the corresponding g values relevant to our centrifuges.

The two applications shown in the video: collecting all the liquid in the tube together in the bottom, and pelleting bacterial cells, are applications that are very often used in molecular labs. For every reaction you set up, you would do a quick spin of 20-30 seconds to ensure that all the liquid is gathered in the bottom of the tube. We have “mini spin” centrifuges for this application. The larger centrifuges are used to pellet cells.

You can read more about centrifugation in the handout that accompanies the first nucleic acid isolation lab. We’ll include a table that shows you rcf (or g) vs rpm for the centrifuges we usually use. This information is in the INTRODUCTION section of the lab handout too- in week 1 of the LabArchives notebook.

D. Simple calculations: examples and practice:

D-1. “V”

“V” refers to a volume and it is a shorthand in protocols that allows us to scale the volumes of the protocol up or down as needed. If we are doing a DNA prep for instance, we could do a very small prep in a small volume or a much larger prep if we are planning to use a lot of DNA. So the protocols are scalable. At certain points, it will say: add 3V ice cold ethanol (for example). This means whatever amount of fluid is already in the tube, you add three times that amount of ethanol. You will see examples of this in the protocols we go over in the lectures and labs.

D-2. “X”

“X” refers to a concentration; it tells you how much more concentrated your stock solution is than the concentration at which you will use it (which is almost always 1X). So if we have a buffer that is labeled 5X, it means it is 5X more concentrated than the concentration you use in the reaction.

So how much of the concentrated (5X) buffer solution do you use in your reaction? It depends on what the total volume of your reaction will be. Suppose it is 100 μl. You want the buffer to be 1X, so you divide the final volume by 5. That is 20 μl. So you will add 20 μl of your concentrated buffer, and the DNA, enzyme and other reagents you need to add, and then sufficient ddH₂O to bring the total volume to 100 μl.

Here are two short questions to practice the calculations. There will be more practice questions on the course website.

A special case of “X” is the loading buffer that we add to DNA or RNA when we run a gel. The loading buffer contains glycerol, to make sure the DNA sinks into the wells of the gel, and one or more dyes that can be seen as the gel runs so you are able to follow the progress of the DNA through the gel. The loading buffer is 6X concentrated for convenience. In this concentration we can easily calculate how much to add to the DNA. For every 5 μl of the DNA sample, we add 1μl of the loading buffer.

E. Basics of gene structure: key points to understand for genetic engineering:

This video shows the process of transcription and translation as a review. You should know what a gene is and how genes are expressed as foundational knowledge for BISC 357.

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