Lab 9 PCR and Gel Electrophoresis

Introduction

The purpose of these labs was to use the polymerase chain reaction (PCR) technique to multiply the isolated DNA from the previous lab 8.  This will allow us to visualize DNA using agarose gel electrophoresis and some colored dye. In order to multiple DNA segments we have to use the PCR technique. This technique generates a large amount of copies DNA, generally of one from something you need for a test using initially a very small sample. For PCR to replicate DNA like the typical natural process, we must supply all of the components normally present for replication in that of a test tube. So we have to force polymerase to synthesize new DNA strands that we need, what can go wrong?! (zombies?). Just add some heat and walla, so we need a thermocycler that will increase the temperature to roughly 95 degrees Celsius. This now denatures the cell allowing us to add whatever we need.

Gel electrophoresis is performed once PCR is completed. This helps us see whats going on. Gel electrophoresis is the technique of separating fragments of DNA based on their size. DNA is injected into a well in an agarose and a dye is used. This will brighten up the data and now will show us the different sizes of DNA present.

Methods and Materials

Obviously, we have to grab those important supplies and fight over what color pipette we want to use. After we are ready to go grab a 25 uL PCR tube following the addition of 2.5 uL 10X Standard Taq Reaction Buffer, 0.5 uL 10 mM dNTPs, 0.5 uL 10 uM forward primer, 0.5 uL 10 uM reverse primer, 2 uL Template DNA, 0.125 uL Taq DNA polymerase, 18.875 uL Nuclease-free water. Now we need some heat so throw it on a thermocycler that will reach 94 degrees Celsius for the next 5 minutes. This will cause a initial denaturation of template DNA.

Now we run 45 cycles of our sample at 94 degrees Celsius for 30 seconds, then 50 degrees Celsius for 30 seconds, 72 degrees Celsius for 30 seconds. Finally, when you’re tired of writing the word Celsius, we need the thermocycler to sit at 72 degrees Celsius for 7 minutes. This will cause changes in temperature that will now allow for Taq Polymerase to copy template DNA strands at an exponential rate. Placed in the freezer at 4 degrees Celsius until it is needed for further experiments.

Now for electrophoresis, we need some squishy agarose gel that is premade by the professor or an assistant. Put gel tray in the gel box, make sure its sealed. Place gel comb where wells are made in the gel for your sample to be placed into. The comb and agar is placed into the electrophoresis. Now once gel is solidified, place 8 uL of ones DNA sample that should be mixed with 2uL of 5X loading dye. Note: Do not pour too much into the gel tray or you will not form clean wells upon solidification!

 This dye is used so that the DNA is visible as it mixes across the agarose. The 10-uL sample is then transferred into a well in the agarose. Mix with the pipette. Now look at the wells. The wells are located by the negative terminal and are completely submerged in a salt solution to allow electricity to flow efficiently through all of the agar. This leads to the power supply which happens to be connected to about 100 volts. Given that the DNA is negatively charged it is attracted to the positive terminal and begins to move toward the other side. The power is turned off when the dye front is near the end of the gel. The distance that the DNA migrates depends on the size of the DNA in question. The larger the strand, the slower it will travel. To get the data, we need to remove the gel and place on a transilluminator. We can take that picture now (selfie).

Results

No results but a picture of what it should look like.

Discussion

The DNA ladder is used to identify the size of the DNA strand, thus also telling us the length of the DNA. A restriction enzyme cuts DNA at a specific location so we can find what DNA will be synthesized for PCR. We can find the location DNA mainly in the nucleus or even the mitochondria which in return form chromosomes. This is how we fit such long strands in such a tight package. Different bands on the gel correspond to different lengths of fragmented DNA strands. The longer the DNA fragments, the slower they will travel through the agar shown above. If multiples show, then we know that the restriction enzyme cut the DNA in multiple places.

Lab 8 DNA isolation

Introduction

            DNA isolation is the process in which we can purify a DNA sample using a combination of physical and chemical methods. In this lab, we focused on a processes called PowerSoil DNA Isolation Kit to layout a specific procedure base to help isolate genomic DNA from the environmental sample one’s heart desires. The kit is used specifically to tackle high humic acid content including difficult soil types such as compost, sediment, and manure.

            We happened to work with a pure culture of E. Coli, a collected activated sludge sample and also a pure culture of a Methylobacterium. We then just isolated the three types of samples to where we can store the Polymerase Chain Reaction (PCR) to use in another lab. This process is a breakthrough for genetic modification in the science world.

Methods/Procedure

            Let us begin with simply taking the designated sample that was given (ours was the Methylobacterium) and add a fourth of a gram to the PowerBead Tubes provided. Gently vortex for maximum mixing power. Next, check for precipitates in your C1 solution, if theres solids then heat at 60 degrees’ Celsius till dissolved. We can now add 60 μL of Solution C1, then

Mix with shaker briefly. Now bust out the vortex and give the O’sample a shaking for 10 minutes at maximum speed. We now can use the best thing ever, The Centrifuge. Centrifuge tubes at 10,000 x g for 30 seconds at room temperature. Now after 30 seconds has passed, transfer the supernatant to a clean 2 mL Collection Tube which should be provided. We can now add 250 μL of Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 minutes. Well, back to the Centrifuge, keeping in mind that the tubes should be at room temperature for 1 minute at 10,000 x g. Avoiding the pellet, transfer up to, but no more than, 600 μl of supernatant to a clean 2 ml Collection Tube which again is provided. Add 200 μl of Solution C3 and vortex briefly. Incubate at 4°C for 5 minutes. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. Avoiding the pellet, transfer up to, but no more than, 750 μl of supernatant into a clean 2 ml in provided Collection Tube. Shake that sample like you’ve never done before to mix Solution C4 before use. Add 1200 μl of Solution C4 to the supernatant and vortex for 5 seconds. Load approximately 675 μl onto a Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Discard the flow through and add an additional 675 μl of supernatant to the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. Load the remaining supernatant onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room temperature. A total of three loads for each sample processed are required. Add 500 μl of Solution C5 and centrifuge at room temperature for 30 seconds at 10,000 x g. Discard the flow through. Centrifuge again at room temperature for 1 minute at 10,000 x g. Carefully place spin filter in a clean 2 mL Collection Tube (provided). Avoid splashing any Solution C5 onto the Spin Filter. Add 100 μl of Solution C6 to the center of the white filter membrane and Centrifuge at room temperature for 30 seconds at 10,000 x g. Discard the Spin Filter. The DNA in the tube is now ready for any downstream application. No further steps are required. If not using immediately, store at -80 degrees’ C.

Results

            We have no results just yet for this lab due to it leading to another set of labs.

Discussion

            We now can see that I typed the word Centrifuge 1 too many times. But this was a interesting lab that was simple but intriguing. We can use the DNA that was isolated for PCR in a later lab.

Lab 7

EES 4102C Wastewater Microbiology

Lab #7: Characterization of Industrial Wastewater

Introduction

                  Wastes brought to landfills are subjected to either groundwater underflow or infiltration from rain water. As water collects throughout the waste in the landfill, it tends to pick up a variety of inorganic and organic compounds. This then flows out of the wastes to accumulate at the bottom of the landfill resulting in contaminated water known to us as leachate. Given that Municipal landfill leachate is high in concentrations of dissolved heavy metals, organic and inorganic compounds.  This all can cause variations in dissolved oxygen, pH, and turbidity. When treatment plants take the leachate, they need to know if the treatment plant can handle the leachate without pre-treatment or if it needs to be pre-treated beforehand.  

Methods and materials

Even though we can look at many aspects of leachate, we are focusing on Specific oxygen uptake rate (SOUR), dissolved oxygen, turbidity, and pH.

Specific Oxygen Uptake Rate (SOUR) was found by first and formost making sure to calibrate the DO meter. We then needed to get about two-300 mL BOD bottle, this was then used to collect varied samples of leachate to wastewater volumes. The volumes can be reported like:

1. 100% leachate and 1% (v/v) leachate:wastewater
2. 100% leachate and 3% (v/v) leachate:wastewater
3. 100% leachate and 5% (v/v) leachate:wastewater
4. 100% leachate and 10% (v/v) leachate:wastewater
5. 100% leachate and 20% (v/v) leachate:wastewater
6. 100% leachate and 50% (v/v) leachate:wastewater
7. 100% leachate and 75% (v/v) leachate:wastewater

Start with the leachate:wastewater mix first! not the 100% leachate. We then filled only half the container as we aerated the sample thoroughly by shaking then place on a stir plate with a stir bar to continue aeration. Now that we have added some aeration we need to fill up the rest of the BOD bottle to just below overflowing with sample. Be sure to immediately insert DO meter and displace enough of the sample to fill top of bottle. This is to isolate contents from the atmosphere. You have to add some movement to the DO probe but very slight to make sure you don’t add too much oxygen back in. After Meter has stabilized, record initial DO (mg/L). Record DO concentration in 2-10 second intervals over a 5 minute period, or until DO becomes stabilized. Then just repeat for given samples.

Now for DO we have to making sure the meter is set to DO (mg/L) mode, rinse the probe with distilled water before each time you make a measurement just to keep contamination at a minimum. Dip the rinsed DO probe into testing solution and stir very slowly! Wait till stable and record the value. Now repeat in triplicates.

Turbidity is a bit tricky with the numbering but very easy to actually do. Turbidity is the measurement that tends to go hand-and-hand with water clarity. To do this you need a handy dandy turbidimeter. Wash the sample bottle in the machine with that of your sample of interest just to minimize contamination. Then fill the vile with your sample and place in the machine. Turn the vile ever so slightly in a 360 degree rotation and pay attention to the smallest number that is displaced. This is to stop the variations in the sample bottle. Repeat in triplicates

We end our experiment with finding pH, This is the total amount of Hydrogen ions in a given sample. The higher the Hydrogen concentrations, the lower the pH. So grab your pH meter and rinse the tip between your measurements. To test the pH, dip the probe into your sample and stir slightly around until giving you a stable number. Now just repeat in triplicates.

Results

Sample calculation shown below:

Sample of 75% 

Has a VSS of 0.11 g/L

Oxygen Uptake Rate of 0.069 mg/L/s

(SOUR) is about 2250 mg/g/hr

Discussion

stay tuned