Gel Electrophoresis

     A gel electrophoresis allows a scientist to analyse DNA. Current is passed through a gel (from - to +), and DNA molecules migrate to the + pole. DNA molecules of different lengths separate, and the shortest segment will move the farthest along the gel. Say we have a plasmid that is cut at one site. This results in a linear DNA fragment, of the same size as the original DNA. Now suppose that we cut it twice. There are now two linear fragments, and one is probably bigger than the other. These two will separate out and will show up as two different bands on the gel. The shorter one will be farther down because it can travel faster.
     If you completed an analysis on the CDS of the plasmid you have digested, you can create a virtual gel that you can later use to see if your RE Digest worked. Go to http://tools.neb.com/NEBcutter2/, and enter your CDS (filter it first using http://www.bioinformatics.org/sms2/filter_dna.html). Then choose circular and hit submit. From there, click on "custom digest" and enter the enzymes you used (in this case, EcoRI and PvuII; your CDS should be the CDS for PGBR22). Then you can click on "view gel" (enter 1% agarose and 1kb DNA marker). If you do this for EcoRI, PvuII, and EcoRI & PvuII, you can see the relative band separations. This is what your actual gel should look like.

To make your gel:

Add 0.7g agarose to 70mL TAE buffer (this will give you 1% agarose). Microwave the solution until it is boiling. Remove the flask from the microwave using insulated gloves and wait for it to cool down, swirling the mixture occasionally. When it is cool enough (you should be able to hold it for 10 seconds), add 0.8uL of ethidium bromide (this is a carcinogen, so be careful!) and swirl it until it is clear. Then, pour the solution in to the casting tray and place the comb (you only need one for an RE digest) in the solution. Wait for the gel to solidify.





To run your gel:

Carefully remove the casting tray and take out the comb. You should be able to see the wells if you look at the gel from a bird's eye view. Place this in the gel electorphoresis apparatus (pictured at the beginning of this post) and fill the chamber up to the fill line with TAE buffer. Then, connect the wires to the corresponding colours on the machine, and set at 105V. If you set the voltage higher than this, the DNA will run faster, but the gel may be prone to breakage (the high voltage makes the solid less stable, and cracks can easily form).

To check your gel:

Carefully remove the gel block from the casting tray, taking caution not to break or crack the gel (this will make viewing it harder), and place it in the UV viewing machine. Flick the switch for the UV light to high, turn it on, and then switch it to low. DO NOT LOOK AT THE UV LIGHT! Find the viewer on the desktop of the computer you are at, and click on it. If you look through the top of the viewing machine, you can see how far your gel has progressed. Use the viewer on the computer to capture a screen shot for future reference. An example is shown below:

However, this does not match the virtual example from the CDS analysis. This suggests that there may be some contamination in the stock solution of the restriction enzymes, or that some of the DNA may have floated from one well to another.

RE Digest

     A Restriction Enzyme digest involves cutting up a piece of circular DNA. Seems simple, right? The problem is, contamination is very common in RE digests, so it is important to know exactly what you are doing, and how everything works.

Background

     Restriction enzymes are isolated from bacteria. They recognize specific sequences of DNA and cut it to produce fragments. Usually, scientists use restriction enzymes to track where the restriction sites are on a certain plasmid or DNA strand. Scientists have isolated enzymes from their bacteria so that they can be used in the lab. In this specific RE digest, we will be using EcoRI and PvuII to cut PGBR22, a plasmid from Montipera efflorescens that codes from a purple fluorescent protein.
     To perform the digestion, obtain a tube each of EcoRI and PvuII, the plasmid you wish to cut (in this case PGBR22), 10X Enzyme buffer (make sure this is a suitable buffer for both enzymes so that they can function at their maximum efficiency), water, and three empty, sterile tubes. In each tube, place 1.5ug of the plasmid (figure out how many uL of the plasmid you need using the concentration) and 2.5uL buffer. In the first tube, put 1uL EcoRI; in the second, 1uL PvuII; and in the third, 0.5uL of each enzyme. Then, calculate how much water you would need to make a total volume of 25uL and add it to each tube. Pipette the sample up and down to mix and spin the sample down in a centrifuge.
     Next, incubate the samples in a water bath at 37 degrees for about 1.5 hours. This will allow the enzymes to do their jobs: cutting the plasmid. After time is up, spin down the samples once more, and place them on a heat block at 80 degrees. This step will denature the enzyme so that it will not function anymore (you do not want the enzyme to keep cutting the already cut plasmid).
     That's all there is to the digestion part. Next, you will have to check if your digestion worked. See the next post on gel electrophoresis!

CDS Analysis

   Once you have figured out which organism's genome your sequence (also referred to as a CDS, a Coding DNA Sequence) matches, you can analyse it. You can take the actual sequence of the organism (this will be more accurate than the one that you received from the DNA sequencing facility) and use it to do a protein BLAST. To do this, first go to http://www.bioinformatics.org/sms2. Enter the actual sequence you obtained from the BLAST and enter it on this website to translate the DNA sequence into a protein. Use all three reading frames. The different reading frames refer to how the DNA sequence can be read. Amino acids are based on three letters within the DNA sequence; therefore, there are three different ways to read the CDS. To find out which reading frame is the most accurate, find the one with the least number of asterisks (*), because each asterisk indicates a stop signal, and the protein sequence with fewer asterisks will have the longest protein. Then, take this sequence (the longest sequence you can obtain) and go back to the BLAST website and click on pBLAST. Enter the sequence, and you will be able to verify that it is from the same organism.


You may be wondering why any of this analysis would be useful. If you take the CDS and insert it into a vector backbone (plasmid), you will be able to express a protein. Protein expression is extremely important, because a scientist can use a certain protein to develop a drug of use to humans.

Submitting DNA to DNA Sequencing Facility

     DNA creates the basis of life. It determines who we are and our traits. Our DNA distinguishes us from plants, and the sequences of our DNA and a plant's DNA are different. For scientists, it is important to be able to sequence DNA to determine the order of nucleotides and to compare the DNA of different organisms. In many cases, knowing the DNA sequence can help make many discoveries. In order to sequence DNA, you will need a purified DNA template, a primer, and a buffer. There are several DNA sequencing facilities, so choose one (usually, they work through colleges or companies).

     The first thing you need to do is to prepare a 1.7mL centrifuge tube. Add 300ng of the template DNA. In order to calculate the amount in ng, use the concentration of the template. If you do not know the concentration, use the nanodrop to find out. Then, choose the primer that corresponds with the DNA template. Add 1uL of 10uM primer OR 4uL of 2.5uM primer. Finally, add nanopure in order to make a final volume of 12uL. When you are done preparing the tube, label it according the the sequencing facility's instructions, and store it as needed.

     When you get your results back, you may need to BLAST (Basic Local Alignment Search Tool) the sequence you received from the facility. A BLAST will tell you if your gene is from the human genome or the genome of another organism, and if so, what organism. This may be able to help you with your research, or it can be used as a check to see if you have the correct DNA. BLASTs are done through NCBI (National Centre for Biotechnology Information). Since your DNA sequence is of nucleic acids, choose nBLAST on the website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Enter the sequence you received from the DNA sequencing facility, and select the option that allows you to search all known genomes. The red indicates matches, so the more red, the better. If you scroll down on the BLAST page, you will be able to see which organisms your sequence matches. Save the information on the BLAST page in order to reference them later if needed.

Making Solutions: Recipes

     One of the key lessons in a lab is making solutions. In research labs, there are usually stock solutions, but it is important to learn how to make each of these solutions yourself. This post gives a list of commonly used solutions in a research lab and how to make them. Be sure to label each solution clearly, with its concentration and pH if needed. Also, include the date.

LB Broth

     The following are needed per litre of LB broth: 10g bacto-tryptone, 5g bacto-yeast extract, and 10g NaCl. If you are making more or less than one litre, you will need to calculate the amount of each ingredient needed. Once you have added the dry ingredients to a bottle, add the amount of water needed (1L if making 1L of LB, 250mL if making 250mL of LB, etc.). Then, shake the bottle to mix it thoroughly. Since LB is usually used as a growth media, it needs to be clean. Place foil over that cap of the bottle and use autoclave tape to hold down the foil. When the autoclave is done, the tape will have changed colour, indicating that the substance has been autoclaved. When you are done, tighten the cap and store the LB broth in a -4 degree Celcius fridge.

Tris Base (pH 7.5, 0.1M)

     Use 2,428g of Tris base to make 200mL of the solution. Pour 80mL of nanopure in a flask and add the tris base to the water. Use a stir bar to dissolve all the dry material. Then, calibrate a pH metre and dip it into the solution, right above the stire bar. Add HCl until the pH reaches 7.5. Add nanopure until you reach the 200mL mark, and store at room temperature.

Ampicillin Solution (50mg/mL)

     To make 1mL of ampicillin, use 50mg of ampicillin powder and place it in a 1.7mL centrifuge tube. Then, add 1mL of water and vortex it to mix the solution. Once all the solid is dissolved, filter sterilize the solution through a 0.22um syringe filter on a 1mL syringe. Dispense all of the solution in a new sterile 1.7mL tube. Wrap the tube in foil and place it in a -20 degree freezer for storage.







TGS running buffer (for protein gels)

     Add 80mL water into a beaker and add 1.51g Tris base, 9.4g glycine, and 0.5g SDS. Stir the solution and add nanopure until you reach the 100mL mark. Store at room temperature.

Nanodrop

     Some experimenters may find a need to know the concentration of a certain substance, usually a plasmid (circular DNA). In order to find out how much DNA you have, you could use a spectrophotometer. However, normal spectrophotometers usually use about 1mL of a sample. In most cases, you do not have this much plasmid. In order to measure the concentration using a small amount of plasmid, you can use a nano spectrophotometer, referred to as Nanodrop.
     If you have access to a nanodrop, install the software on your computer, and click on ND1000 to open the software. Select the nucleic acid setting if you are measuring DNA . Make sure DNA-50 is selected in the top right. Wipe both the upper and lower terminals with nanopure to clean the terminals. Then, initialize the instrument with water by placing 2uL of nanopure water on the sample pedestal, lowering the arm, and clicking okay. Then clean the pedestal to remove the water. Next, blank the insturment by placing 2uL of your elution buffer (ex. TE, Tris HCL, etc.) on the pedestal and clicking blank. It is important to blank the instrument using the buffer your DNA is in to account for any background absorbance from the buffer. Then, clean off the pedestal again. Finally, measure your sample by placing 2uL of it on the pedestal and clicking measure. The output is in ng/uL. Record the 260/280 value and the 260/230 value. These ratios will give you the purity of the DNA if you need it. Print out your graph.

     To be more accurate, take a second measurement and average the readings. The concentration of plasmids are usually abbreviated using the symbol λ, which means ug/uL. If your value is 1.5λ, then that is equal to 1.5ug/uL, which is equal to 1500ng/uL.

The Use of Micropipettes

     Scientists use micropipettes when dealing with very small amounts of liquid. The samples micropipettes can take range from 0.01 uL (microliters, or 10^-6 of a liter) to about 1000uL (1mL). The following table shows different pipette sizes and their tips:

     In addition to these sizes, there is a P-2, which pipettes from 0.01uL to about 2uL for more precise measurements. It is important to know how to use a micropipette correctly, because measurement is key when it comes to research. When working with materials that can be easily contaminated, make sure you change tips each time to reduce cross contamination.

     The first thing to remember when learning how to use a micropipette is to hold it properly. Grip the pipette so that the ledge rests on your index finger, and you can press down the plunger with your thumb. Remember, the plunger is different from the tip eject button. If you press this one by accident, your tip will be pushed off the pipette. To adjust the volume you want to pipette, turn the black knob at the top so that it does not make a right angle. Then you can twist the plunger portion of the pipette, and you will see the numbers on the side change. This number indicates the amount of liquid you are pipetting. When you are done, twist the black knob back to lock the measurement. To pipette, push the plunger until you feel an initial stop (not all the way down), and then insert the tip into the liquid you wish to pipette. Let go of the plunger completely. Then push the plunger completely down to the last stop into whatever container you want the liquid in. That's all there is to it!

Safety Comes First

     The first pieces of equipment a researcher should familiarize himself or herself with is the safety equipment. Gloves, goggles, and lab coats are all required to maintain a safe working environment. Ethanol should be used to clean any surfaces that are going to be used. Know where the eye wash, emergency shower, fire extinguisher, fire blanket, and phone are, and how to work all of them. Make sure you know where biohazard containers are placed, and what goes in them (usually anything that contains DNA or possibly harmful material).

    http://www.dartmouth.edu/~chemlab/info/safety/equipment.html

     Most labs will have a designated area for gel electrophoresis, because of the use of ethidium bromide, a carcinogen. Anything used in this area should stay in this area to reduce contamination.

 





   
    Also, know how to use a Bunsen burner, and make sure it is turned on when you are working with bacteria, in order to keep the general area sterile.

 





     After you are done with an experiment, know how to clean up after yourself. Clean the surface you used with ethanol, and spray and beakers with alcanox, or bleach, if needed. Make sure all glassware is completely clean.

     Sometimes, biological waste is cleared using an autoclave. Know where the nearest autoclave is, and how to use it. Autoclaves function at high temperatures, so be sure to use the designated gloves when working with them.

     Well, there you have it! Lab safety in a nutshell.

The Research Process: An Introduction

     Research is the basis of all science, and the first step to any discovery. I will be covering drug-based discoveries in this blog. Throughout the year, I will experiment with a target protein, which I will determine later. More details on this target will be given, but the main purpose of this blog is to expose the reader to a research method and educate him or her about the scientific process.

     In order to begin research, the first thing that must be done is that the researcher must become familiar with the equipment he or she is working with, and should be able to perform several basic experiments without hesitation. This is an important step in the creation of a researcher, because without the basic understanding of how scientific equipment works, a researcher will not be able to understand the basis of his or her research.