CE 742 Advanced Topics in Environmental Engineering Lab Manual
Overview:
Each team has been assigned so that at least one partner has some experience with molecular techniques. Each team should choose a sample relevant to Environmental Engineering which they will focus on for the semester. During the course of the semester you will qualitatively and quantitatively describe the bacterial composition of this sample.
General Guidelines in the Lab:
Proper attire:
Always wear gloves in the lab! This is both for your protection, and also to protect the samples from contamination from your skin. Remember, DNA is everywhere, you want to make sure you are studying the DNA from your sample, and not random environmental DNA.
A lab coat or other lab smock is recommended to protect your cloths from spills. You may keep this in the lab for use during lab classes. Also remember to wear closed-toed shoes and long pants or skirts which protect your legs.
Waste Disposal:
Most waste generated in this lab can be disposed of in the regular waste bin. However, there are some exceptions noted below. These should be disposed of in the appropriate marked container for hazardous waste collection.
Hazardous Chemicals:
Ethidium Bromide (EtBr): This is used to stain gels and is a suspect carcinogen.
Formamide: This is used in small quantities in PCR and also in the making of the DGGE gel in order to lower the melting temperature of DNA.
Acrylamide: This is the main ingredient in the DGGE gel, and is a known neurotoxin. We will only use liquid acrylamide, which eliminates hazards associated with inhaling the powder form.
Using the pipeters:
Pipeters are expensive tools that must be cared for properly. Each group has 3 pipeters with 3 volumes: 0.5-10 ml, 10-100 ml, and 100-1000 ml. Always choose the appropriate pipeter for the appropriate volume. Set the volume on the pipeter and then take a tip with the pipeter. When taking sample, release the button slowly to draw sample into the tip. If you release the button too quickly, the volume will not be accurate, and may splash inside the pipeter. Always hold pipeter vertically, turning the pipeter sideways with sample in the tip will contaminate the pipeter and your sample. Finally, after taking the sample, check to make sure there are no bubbles in the tip before dispensing the sample. Dispense of the tip and use a fresh tip for each sample.
Team Assignments:
Group 1
Luciana Pererya, Mary Beth Talty, and Chris Messersmith
Group 2
Ruoting Pei and Mustafa Yarkin
Group 3
Anurita and Matt Stephens
Group 4
Matt Hoelscher and Winnie Lin
Lab I: DNA Extraction and Quantification
Objective:
You will extract DNA from the sample of your choice using the QBiogene FastDNA SpinKit for Soil. This method employs physical disruption of the cells (bead-beating) followed by physical binding the DNA to a silica matrix, washing with an Ethanol based solution, and eluting in purified water. Extraction will be verified by agarose gel electrophoresis and quantified by spectrophotometry.
Procedure:
Selection of Sample:
Each team should select a sample of Environmental Engineering relevance for DNA extraction. Examples include: bacterial cultures (relatively easy to extract), soil (relatively more difficult), sediment, or sludge. You may also choose a sample relevant to your graduate research. Make sure this sample is of interest to you, you will be studying this sample the rest of the semester.
Bring with you enough sample to perform the extraction in duplicate. For soil or other solid sample, 0.5 g per sample is required. For culture, 1 ml is typically sufficient, but you will need to centrifuge it first, pour off the supernatant, and transfer the pellet to the extraction tube. If you are unsure, ask the instructor for advice on what sample and how much to bring.
Agarose Gel Preparation:
First you will prepare an agarose gel. You will need this after DNA extraction to verify that it worked. You will also use this procedure throughout the semester to visualize DNA.
1.) Set up the casting tray in the gel caster. Place the comb with the appropriate number of wells in place at one extreme end of the gel tray.
2.) A 2.5% solution of agarose in 1X TAE buffer (already prepared and melted at 55ºC). *Caution! Wear gloves!:This solution contains 3 ml per 100 ml of ethidium bromide, a DNA intercolating agent, which binds to DNA and fluoresces under UV light. This will allow you to visualize the DNA later. This agent is also a suspect carcinogen, and should not be allowed to contact the skin.
3.) Take this solution and pour into the gel tray- fill until about ¼ cm from the top of the space in the comb. If any bubbles form while pouring the gel, you may pop them or push them to the side using a pipet tip.
4.) Set aside and allow the gel to solidify.
DNA Extraction:
A. Disrupting the Cells:
1.) Do a duplicate extraction of your sample. Weigh the empty matrix tubes and record the weight. Add your sample to the “matrix tubes,” and record the volume with sample. Use a sterile instrument (such as a spatula or pipet tip) to transfer the sample into the tubes.
2.) Add 978 microliters of phosphate buffer and 122 microliters of MT Buffer to the matrix tube using the 100-1000 microliter pipeter.
3.) Place cap on tightly and secure tubes in Bead-beater. Make sure that samples are balanced, as you would in a centrifuge. Place setting on “Homogenize”, and run for 3 minutes. Note: because of the force exerted in the bead-beating process, it is recommended that you place the bead-beater on the floor during sample processing.
4.) After bead-beating, centrifuge sample for 1 minute on highest setting (making sure samples are balanced).
B. Binding the DNA:
5.) Transfer the supernatant (the liquid forming the top layer) to a fresh 1.5 ml centrifuge tube. Add 250 ml of PPS (protein precipitating solution). Mix by inverting the tubes 10 times, then centrifuge on the highest setting for 5 minutes.
6.) While centrifuging, label one 15 ml centrifuge tube (with blue cap) and add 1 ml of Binding Matrix (make sure to shake the binding matrix prior to adding). After centrifuging, transfer the supernatant to a 15 ml centrifuge tube, taking care not to disturb the pellet. Swirl the sample gently to mix. Do this several times to maintain the sample in suspension for 2 minutes.
7.) Set the 15 ml tubes aside and allow the binding matrix to settle. Once settled, remove 0.5ml of the supernatant with the pipet tip and discard, taking care not to remove any settled binding matrix.
8.) Resuspend the sample by swirling and transfer 600 microliters into a tube with a spin filter. (make sure to label these tubes appropriately). Centrifuge the tube with the spin filter for 1 minute. The DNA should stay bound to the matrix, and the remaining solution will come down into the bottom of the tube. After centrifuging, open the tube, take the spin filter out with one hand, and with the other, discard the flow-through in the bottom of the tube. Replace the spin filter and add 600 more microliters of the suspended binding matrix. Centrifuge, discard flow-through, and repeat with remaining sample.
C. Washing the DNA:
9.) Add 500 ml of SEWS-M to the spin filter. Centrifuge and discard flow through. After discarding flow-through, centrifuge empty tube for 2 minutes to “dry” the spin filter.
10.) Transfer the spin filter containing the washed binding matrix to a fresh catch tube. Allow to sit for an additional 5 minutes with the lid open to further dry the binding matrix.
11.) Add 50 ml of DES (ultrapure water, DNA and pyrogen-free) and stir the binding matrix gently with the pipet tip. Be careful not to put a hole in the spin filter while stirring.
12.) Let incubate 2 minutes and then centrifuge for 1 minute on high. Check to make sure that the flow-through is clean, and none of the binding matrix passed through the filter (If this happens, transfer the sample to a fresh tube, being careful not to transfer any of the binding matrix.
13.) DNA is now extracted! Be sure to label this final tube appropriately for storage (Name, date, sample identirication). Check product on an agarose gel and store sample at -20ºC.
Loading and Running an Agarose gel:
A. Setting up the Electrophoresis Unit
1.) Carefully remove the comb from the solidified gel by pulling the comb slowly upwards.
2.) Prepare 1 L of 1X TAE buffer solution by diluting the 50X concentrated TAE buffer with DI water (eg, 2 ml of 50X TAE per 100 ml solution = 1 X TAE).
3.) Turn the gel tray containing the solidified gel so that the wells are closest to the negative (black) electrode. [DNA has a negative charge, so will travel towards the positive (black) electrode once potential is applied.]
4.) Pour the 1X TAE solution over the gel so that it fills the wells in the gel, and also fills the electrophoresis chamber flush up until the level of the buffer is flush with the top of the gel.
B. Preparing the Samples and Loading the Gel
5.) Now cut a piece of parafilm (about 2 inch by 4 inch should be sufficient) and place on the lab bench, parafilm side up.
6.) Using the 0.5-10 ml pipeter, place four 2 ml “dots” of blue loading dye on the parafilm (one dot for each of the samples that were extracted, and one dot for each of the molecular weight standards).
7.) Now take 3 ml of the first molecular weight marker, and add to the first blue dot. Mix by pipeting up and down. Once mixed, take the sample and dye mixture back up in the tip.
8.) Load this sample into the first well. Do this by placing the tip gently about midway into the well, and slowly releasing the mixture into the well (Careful! Do not pierce the bottom of the well!). The blue loading dye contains glycerol, which makes the sample sink to the bottom of the well, and helps prevent the samples from coming out of the wells and cross-contaminating other wells.
9.) Repeat this procedure with the two DNA extracts, and finally with the second molecular weight marker.
C. Running and Visualizing the Gel:
10.) Once the gel is loaded, place the cover on the electrophoresis unit (red to red, black to black) and plug in the leads to the power supply (red to red, black to black). Set the voltage on 120 v and press “run.” If you are running two gels from the same power supply, assuming that the resistance of each gel is the same, set the voltage on 240 v. Run until you see the blue dye move about halfway down the gel. At this time, turn off the voltage to the gel.
11.) In order to “see” the DNA, you need to look at it 1. 2. under UV light. Take the gel over to the hand-held UV lamp. Make sure that you are wearing the UV protection shield over your face, and that any bare skin is covered completely by gloves and your sleeves. Turn on the lamp and hold over the gel. If your extraction is successful, you should see something similar to Fig. 1. On your gel you will have two molecular weight standards on either side of your samples.
Fig. 1: Example of DNA extract run on agarose gel and visualized under UV light. 1.) molecular weight marker or “ladder” with top band of DNA = 1500 bp, brightest band (middle) = 500 bp, and bottom band = 100 bp. 2.) Extract of genomic DNA from E. coli.
Quantifying the DNA:
The brightness of the band of the extracted DNA on the agarose gel should give you an indication of the yield of your extracted DNA. Now we will use spectrophotometry to better quantify this yield. You may want to get this part started while you are running the agarose gel.
1.) Warm the lamp on the spectrophotometer for 30 minutes prior to use.
2.) Make a 1:100 dilution of your DNA extract, with a final volume of 500 microliters (eg. 5 microliters of DNA extract added to 495 microliters of D.I. water). You can do this in a microcentrifuge tube. You may use regular D.I. water to do the dilution (it is not necessary to use the ultrapure water, since you will throw away this sample after measuring it).
3.) Once the lamp is warmed-up, Fill the quartz cuvette with the same water you used for the dilution. Zero the instrument. Note: Handle the quartz cuvette carefully, it is expensive! Only wipe the surface of the cuvette with kimwipes, other materials may cause scratches.
4.) Once the instrument is zeroed, check the samples. Rinse out the cuvette with D.I. water, “tap” dry on a kimwipe, and transfer the sample to the cuvette using the pipeter.
5.) You will determine the absorbance at two wavelengths: 260 nm (DNA) and 280 nm (protein). A high ratio of 260/280 indicates that the sample is relatively pure with respect to protein contamination.
6.) Use the following formula to determine the concentration of DNA in your sample:
To calculate the concentration of genomic DNA in the dilution:
A260 * 50 ng / ml = x ng / ml
To calculate the concentration of genomic DNA in the extract before dilution:
x ng / ml * (500 ml / 5 ml) = y ng / ml
To calculate the total mass of genomic DNA in the extract: