A pipette  is a tool used for measuring and transferring liquids. There are several different types for different applications. Below you can see a transfer pipet (Image A),  micropipetters (Images B and C), and serological pipettes (Images D, E and F). The transfer pipette (Image A) is usually only used for transfer of somewhat small volumes when precise measurement is not needed, such as adding a few drops of base as you adjust the pH of a solution that you are making. The micropipette (Image B) is one of the most commonly used tools in the lab and is used when very precise, small volume measurement is needed. We have a number of micropipetts, each with a specific volume range (Image C). The ranges include 0.5 μl (microliters) to 2.5 μl, 2 μl to 20 μl, 20 μls to 200 μl, and 200 μl to 1000 μl. If one needs to measure and transfer volumes between 1 mL and 10 mL, serological pipette might be used. There are several devices which can be used with serological pipettes, including  those seen in images D, E, and F. For measuring and transferring larger volumes than this we would use a graduated cylinder.

Here is a great video about using a micropipetter from NC Community Colleges:

A centrifuge is a device that is used to separate components of a mixture by their density. A centrifuge has a rotor which is used to hold tubes or bottles of the mixture. The rotor is seated on a spindle, and spun at very high speeds. It is critical that the tubes in the rotor are balanced, or the spindle can break, turning the rotor into a dangerous projectile. We have three different types of centrifuges in the lab. We use the microcentrifuge (Image A) with 1.5 mL tubes. The table top centrifuge is often used when we prepare our protoplasts (Image B), and has rotors to hold 15 mL conical tubes and 50 mL conical tubes. The refrigerated centrifuge (Image C) has rotors for tubes ranging from 15 ml to 50 ml and bottles ranging from 250 ml to 1 liter. We use it to collect bacteria from liquid cultures and to collect organelles, membranes or proteins from homogenized tissues.

Here are some important tips about centrifuge use:

Polymerase Chain Reaction (PCR) is a powerful tool that allows us to make many copies of specific segments of DNA. Here is a nice animation and description of the process from the DNA Learning Center: There are typically five required components for each PCR reaction: buffer, DNA, primers, dNTPs, and TAQ Polymerse. These  components are placed into a PCR reaction tube (Image A). It is critical to keep the components chilled prior to beginning the reaction series  to prevent “mispriming”, which occurs when the primers anneal non-specifically and synthesis starts at room temperature. When all the reaction tubes are prepared and the PCR machine has been programmed, the tubes are put into place (See image B) and the lid is carefully closed (Image C). The lid is heated to prevent condensation from forming inside the reaction tube lid. If this occurred, the concentration of other reaction components in the bottom of the reaction tube would change, and this could disrupt the activity of the polymerase. The program displayed in Image D has 35 cycles: an 8 second denaturing step at 98 degrees C, 25 second annealing step at 60 degrees C, and a one minute elongation step at 72 degrees C. The high temperature of the denaturing step breaks the hydrogen bond for the DNA’s double helix, causing it to become single stranded. In the annealing phase, the primers will bind to the single stranded DNA where complementary base pairs are “found”. These provide the starting point (an available 3″-OH group) for DNA replication, so when the temperature is raised to 72 degrees C, the TAQ Polymerase begins building a new DNA molecule based on the template found by the primers.

 There are lots of applications for PCR. One technique that we are using in the lab helps us to find unknown CESA genes using primers developed using sequences of known CESA genes from other species. If we are able to amplify DNA using our primers, is can lead us to discover similar genes in other organisms.

Gel electrophoresis is a technique that we use to separate mixtures of nucleic acids (and sometimes proteins) by their size. Here is video produced by Biorad that does a nice job describing this procedure:

https://www.youtube.com/watch?v=vq759wKCCU

Below are some of the materials that we use for DNA gel electrophoresis. First the gel is created by combining buffer and agarose. This is heated in the microwave to melt the agarose. It is important to measure the mass of the flask containing your buffer and agarose before heating so that after heating the correct amount of distilled water can be added back to the flask to account for that which evaporated during heating. Ethidium Bromide is added to the gel immediately before casting. Once this is added, the contents of the flask are carefully poured into the casting tray, avoiding the creation of bubbles (Image A). The gel will be cool, firm, and ready for use after about 20 minutes. While the gel is cooling, it is a good time to draw a chart in your lab notebook so you know exactly what will be in each lane of your gel (Image F). Often the first lane is used for the “DNA Ladder,” a sample with segments of DNA of known sizes for comparison. You can also begin to get your DNA samples prepared by mixing with appropriate volume of sample dye (Image B). This dye mix helps us to track the progress of DNA separation in the gel, so that it is not run too long resulting in the DNA falling right out of the end of the gel and into the buffer. Once cooled, the gel is placed into the gel box and covered with running buffer.  Samples are loaded into their respective wells, the gel box is covered, and the electrodes are attached to the power supply (Images C and D). Gloves must be worn when handling the gel, as it contains ethidium bromide. Also, one you have handled a gel with a pair of gloves, you must take care not to touch other items in the lab. After the gel has run for the designated time, carefully place the gel on the UV transilluminator inside the imaging system (Image E). Remove a glove and use that hand for operating the computer to record and image of the gel (Image G).

When working with cultures, we use a laminar flow hood to reduce the likelihood that contaminants like bacteria and fungi will fall from the air or off of our hands or clothing into our cultures and contaminate them.

Before working in the hood, familiarize yourself with sterile or aseptic technique. You tube has a lot of helpful videos offering tips and advice. Here are a couple:

This is a nice review of aseptic technique from Invitrogen. Keep in mind, though, that they are demonstrating in a “Biosafety Cabinet,” but in our lab we are currently using a laminar flow hood: http://youtu.be/4mKhULnxqcw This video shows the difference between airflow patterns in a biosafety cabinet and a vertical laminar flow cabinet. Ours, however is a horizontal flow cabinet. As such, one would never use our cabinet for handling anything pathogenic as the air flow is directed at the worker and is not intended for containment. The intention with the horizontal flow cabinet is to prevent materials from falling into cultures. It is therefore appropriate for use in culturing plants and non-pathogenic fungi.

Consistent and correct preparation of culture medium is absolutely critical. Documentation and tracking of batch numbers can be very helpful in trouble shooting any culture challenges that may arise.  Here is a short video from PhytoTech Labs with some good advice on media preparation:

In our lab we have a designated area for media preparation where the balance, pH meter, type II water system, refrigerator, freezer, fume hood, and glassware cabinet are all located.

Here is a great animation to help understand western blots:

http://ow.ly/uKBhh