This lab is a short course in basic skills that are needed in BIO 401 lab. There are three things that will be important in the lab that your should understand before starting any procedures; how to dispose of biohazards, how to make dilutions, and how to use the pipettors.
Biohazards
One of the greatest fears of the public concerning molecular biology is that a scientist will create something in the lab that will accidentally escape and take over the world. While this is, for the most part, irrational (if it happens, it won't be an accident - see "Twelve Monkeys"), there is still the possibility that an accident could occur if proper decontamination procedures aren't followed. We won't be working with any dangerous biologicals, but it's best to learn the techniques and stick to them, just in case.
The most important thing to remember is that anything that touches a solution of bacteria or DNA is considered contaminated. Put pipette tips, plastic tubes, agar plates and anything else that has contacted these things into the special waste container on your bench and then dispose of them in an orange BIOHAZARDS bag. I will autoclave this material later and dispose of it properly.
Dilutions
One thing I constantly hear from students that disturbs me is "I can't do dilutions." It's as if it is a regrettable, yet unavoidable circumstance that must be accepted. The truth of the matter is, if you leave here without the ability to make an accurate dilution, you won't be worth anything in a lab. It's actually a very simple procedure; it just takes practice. There's only one equation to remember:
Another thing to remember is the conversions between prefixes (representing 3 orders of magnitude) in the metric system. Here is a refresher, from very big to very small:
tera (T) giga (G) mega (M) kilo (K) unit (e.g. grams) 10E12 10E9 10E6 10E3 1 milli (m) micro (u) nano (n) pico (p) femto (f) 10E-3 10E-6 10E-9 10E-12 10E-15
If you don't know these already, you should know between giga and pico.
Practice Exercises
Here are some dilution problems that you should be able to do. You will be given identical problems on next week's quiz, so be sure you understand them.
1.) Make 25 ml of 20% ethanol from a 95% ethanol stock.
2.) Make 500 ml of 1X SSC from a 20X SSC stock. (Remember, it doesn't matter what the units are or what the chemicals are. The calculation is the same.)
3.) Make 20 ul of 1X restriction enzyme buffer from a 10X stock.
4.) Make 20 ml of 0.5% NaCl from a 10% stock.
5.) Make 100 ml of a solution that is 5 mM MgCl2, 5 mM CaCl2 and 125 mM KCl from 1 M stocks of each of these chemicals.
Do these problems first, then check your work with the answers.
If you have problems with these dilutions, please come see me and we'll work on them together. You will have a few dilution problems on your first quiz and one on each quiz after that.
The Pipettor
The pipettors that we will be using are accurate measuring devices for very small quantities of liquid. This will be important in our work because we will often be making solutions with four or five components whose final volume is 20 ul (0.020 ml) which smaller than a drop of water.
As stated above, these pipettors can be very accurate if used properly. If used improperly, they will be all over the place. Pipetting will be the basis of almost all of the experimental work that we do, so it is important to develop good technique in order to get good results. There are a number of rules that must be followed for good technique:
1. Set the volume gauge carefully. Be sure that it is set on the desired volume.
2. Put a new tip on the pipettor. Always start with a new tip (unless you are doing repeated dispensing of the same solution).
3. Rinse the inside of the pipette tip with the solution that you will be dispensing. This is critical to getting accurate results. Push the plunger down, put the tip into the solution, and slowly pull the solution up and down into the tip. You should repeat this 8 - 10 times before taking the sample. Then empty any liquid out of the tip.
4. When taking your sample from the solution, be sure to pull the solution up into the tip very slowly. If you pull up too fast, the liquid will jump up and you will get air in the tip. This totally ruins any chance of making an accurate measurement and will probably contaminate the pipettor itself.
Another key to getting accurate results is using the proper pipettor for the volume you need. There will be three different pipettors available to you; the P-1000, P-200 and the P-20. Each is clearly marked on the top. You should use the following guide in the selection of the proper pipette:
For volumes from 1000 ul to 200 ul -------> P-1000
For volumes from 200 ul to 20 ul -------> P-200
For volumes from 20.0 ul to 0.5 ul -------> P-20
If you use the pipettors outside these ranges, their accuracy drops of very quickly. A couple of personal notes. For volumes between 1.0 ml and 5.0 ml, I find that it is easier to use a P-1000 multiple times to make up the volume than to use a graduated pipette with a pipette bulb. For instance, if I had to pipette a volume of 3.5 ml, I would set my P-1000 on 1000 ul and pipette 3 times, then set it at 500 ul and pipette once . Or, more likely, I would set my P-1000 on 0.875 ml and pipette this volume four times. Also, the P-20's that we are using are set so that they won't go below 2.0 ul. Often volumes as small as 0.5 ul are needed. To pipette these volumes, you need a little experience in gauging what 0.5 ul of liquid will look like in the disposable tip. I will show you this technique in class.
Testing the Accuracy of Pipetting
In this first lab, you will practice pipetting to develop good technique. To do this we will be pipetting water and weighing the volume to determine accuracy. Since the density of water is 1.0 g / ml, the mass of the volume of water pipetted in grams is equal to its volume in ml. This simple conversion will be used to determine the accuracy of your pipetting.
Procedure:
1.) Put a piece of weigh paper on the balance and tare it so that the scale reads 0.000 g.
2.) Set your P-1000 for 1000 ul and put a tip on it. Push down the plunger, put it in the water and pull up slowly. Put this amount of water on the scale and weight it. Record this weight on a piece of paper.
3.) Dump the water off the weight paper and re-tare it to 0.000 g.
4.) Using the same pipette, rinse the tip with water. Do this ten times slowly. Now push down the plunger and allow it to jump up quickly. Weigh this amount of water and record it.
5.) Dump the water and re-tare the balance. This time pull the water into the pipette tip very slowly. Weigh this amount of water. If this doesn't weigh 1.000 (+/- 0.005) g, then repeat this slowly until you get this degree of accuracy.
6.) Repeat step five with two other volumes. Good volumes to use are 0.500 ml and 0.200 ml.
7.) Do step five using the P-200 at 200 ul, 100 ul, and 25 ul, until you get accurate results (< 0.5% error).
8.) Do step five using the P-20 at 20.0 ul, 10.0 ul, and 5.0 ul, until you get accurate results.
If you have questions please send me a message at my Email address
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