DNA Extraction
DNA extraction (salt ethanol technique) was practiced on a variety of different plant tissues- including strawberry (Fragaria x ananassa), kiwi (Apteryx australis), onion (Allium cepa), common store-bought mushrooms (Agaricus bisporus) and chicken (Gallus gallus) liver.
Strawberries consistently give the highest DNA yield (based on visible stranded 'chunks' of DNA that can be spooled out of ethanol layer). We would have liked to quantify our DNA & get an idea of protein contamination but our Spectrophotometer (LoggerPro SpectroVis Plus Spectrophotometer) only goes down to 380 nm. For estimating DNA concentrations and protein contamination, we need to read absorbances at both 260 nm and 280 nm which are beyond what our LoggerPro SpectroVis can measure.
A few things for our wish list of equipment: NanoDrop Spectrophotometer and micro-centrufuge!
We used the following protocol from the Learn Genetics website (University of Utah)-
1. Obtain tissue from your DNA source (about 125ml or 1/2 cup).
Add a large pinch of table salt (about 1g or 1/4 teaspoon).
Add twice as much warm water as the DNA source (about 250ml or 1 cup).
Blend on high for 15 seconds in blender.
2. Pour 'cell soup' through a strainer (cloth/coffee filter/filter paper) into another container.
Measure 12ml of this soup into a small beaker and add 2ml of liquid detergent and swirl to mix.
Let the mixture sit for 5-10 minutes in a container of hot tap water (60°-65°C), occasionally swirling gently. Remove the mixture from the hot tap water and allow it to cool to room temperature.
3. Add several drops of contact lens cleaning solution (Rinso)
4. Pour your mixture into a test tube until it is 1/3 full. Tilt the test tube and slowly pour ice-cold alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the cell mixture. Do not mix the two layers together. Pour until you have about the same amount of alcohol in the tube as cell mixture.
DNA precipitates as a white stringy/snotty film at the water-alcohol interface and eventually will rise into the alcohol layer from the mixture layer. Allow your test tube to sit for several minutes. The clearer your DNA is, the fewer impurities you have. You can usually get more DNA to precipitate from the solution by using a DNA-collecting tool (such as a glass or paper clip hook) to gently lift the water solution up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate.
When you have an acceptable amount of DNA, it can be ‘spooled’ by rotating your collecting tool and then transferred into a clean tube. Leave the tube uncapped until the ethanol has evaporated. Your DNA can be stored dry or water/buffer can be added. You will need to agitate the tube gently to dissolve the DNA- this may take some time. DNA can be stored in the fridge.
Strawberries consistently give the highest DNA yield (based on visible stranded 'chunks' of DNA that can be spooled out of ethanol layer). We would have liked to quantify our DNA & get an idea of protein contamination but our Spectrophotometer (LoggerPro SpectroVis Plus Spectrophotometer) only goes down to 380 nm. For estimating DNA concentrations and protein contamination, we need to read absorbances at both 260 nm and 280 nm which are beyond what our LoggerPro SpectroVis can measure.
A few things for our wish list of equipment: NanoDrop Spectrophotometer and micro-centrufuge!
We used the following protocol from the Learn Genetics website (University of Utah)-
1. Obtain tissue from your DNA source (about 125ml or 1/2 cup).
Add a large pinch of table salt (about 1g or 1/4 teaspoon).
Add twice as much warm water as the DNA source (about 250ml or 1 cup).
Blend on high for 15 seconds in blender.
2. Pour 'cell soup' through a strainer (cloth/coffee filter/filter paper) into another container.
Measure 12ml of this soup into a small beaker and add 2ml of liquid detergent and swirl to mix.
Let the mixture sit for 5-10 minutes in a container of hot tap water (60°-65°C), occasionally swirling gently. Remove the mixture from the hot tap water and allow it to cool to room temperature.
3. Add several drops of contact lens cleaning solution (Rinso)
4. Pour your mixture into a test tube until it is 1/3 full. Tilt the test tube and slowly pour ice-cold alcohol (70-95% isopropyl or ethyl alcohol) into the tube down the side so that it forms a layer on top of the cell mixture. Do not mix the two layers together. Pour until you have about the same amount of alcohol in the tube as cell mixture.
DNA precipitates as a white stringy/snotty film at the water-alcohol interface and eventually will rise into the alcohol layer from the mixture layer. Allow your test tube to sit for several minutes. The clearer your DNA is, the fewer impurities you have. You can usually get more DNA to precipitate from the solution by using a DNA-collecting tool (such as a glass or paper clip hook) to gently lift the water solution up into the alcohol. This allows more DNA to come in contact with the alcohol and precipitate.
When you have an acceptable amount of DNA, it can be ‘spooled’ by rotating your collecting tool and then transferred into a clean tube. Leave the tube uncapped until the ethanol has evaporated. Your DNA can be stored dry or water/buffer can be added. You will need to agitate the tube gently to dissolve the DNA- this may take some time. DNA can be stored in the fridge.
E. coli Cultivation- Aseptic Transfer & Streak Plates
Escherichia coli (E. coli) is a motile Gram-negative bacillus that is commonly found in the intestines of humans and other animals. There are different strains of E. coli which can cause severe food poisoning in humans. A gram - negative bacillus is a type of bacteria which retains a crystal violet dye and stain dark violet/purple with gram stain when it is washed with alcohol and water.
This time we have learnt how to create new bacteria cultures from a preexisting cultures. First we have to prepare an agar solution and set it in a sterilised Petri dish. Agar is a gelatinous like substance derived from seaweed. It contains a wide range of nutrients which are required for bacteria growth and reproduction, which is why it is an ideal and the most common culture base.
Sterilising equipment before this process is crucial in order to ensure that the bacteria culture is not contaminated by impurities. Petri dishes and Inoculation loops should be sterilised in a pressure cooker in order to kill any organisms that are present on the equipment. By heating the equipment up under such high pressure, it eradicates micro-organisms more efficiently as they will die due to the high temperature.
There are limitations with using the inoculation loop to spread the bacteria sample onto the new petri dish. One thing you have to make sure is that your agar is set and completely firm, or else it will be difficult for you to spread it and you will end up breaking the agar layer. This is partly due to the low quantity of agar used in our first batch of petri dishes. The plates we made after that were harder & easier to transfer bacteria from & to with an inoculation loop.
Using the glass beads to spread the bacteria does indeed give you a higher chance of attaining a more even layer of bacteria, however it does not allow you to create individual colonies which can be achieved by using the inoculation tube.
This time we have learnt how to create new bacteria cultures from a preexisting cultures. First we have to prepare an agar solution and set it in a sterilised Petri dish. Agar is a gelatinous like substance derived from seaweed. It contains a wide range of nutrients which are required for bacteria growth and reproduction, which is why it is an ideal and the most common culture base.
Sterilising equipment before this process is crucial in order to ensure that the bacteria culture is not contaminated by impurities. Petri dishes and Inoculation loops should be sterilised in a pressure cooker in order to kill any organisms that are present on the equipment. By heating the equipment up under such high pressure, it eradicates micro-organisms more efficiently as they will die due to the high temperature.
- Grab samples of bacteria from the preexisting culture using the inoculation loop.
- Spread the bacteria on the petri dish with set agar. You can either use the inoculation loop to spread it or glass beads to roll the bacteria evenly on the surface of the agar.
- Close the petri dish and leave it inside an incubator for the bacteria to grow and reproduce.
There are limitations with using the inoculation loop to spread the bacteria sample onto the new petri dish. One thing you have to make sure is that your agar is set and completely firm, or else it will be difficult for you to spread it and you will end up breaking the agar layer. This is partly due to the low quantity of agar used in our first batch of petri dishes. The plates we made after that were harder & easier to transfer bacteria from & to with an inoculation loop.
Using the glass beads to spread the bacteria does indeed give you a higher chance of attaining a more even layer of bacteria, however it does not allow you to create individual colonies which can be achieved by using the inoculation tube.
Streak PlatesThe goal of making a streak plate is to isolate individual bacterial colonies that arose from individual cells. While at first we had a bit of trouble with our agar (too soft) we eventually got it right. While we are able to transfer our bacteria from plate to plate without contamination but are still having troubles making good streak plates (see images). This might be a problem in completing our first bacterial transformations. Below are some images of our 'dream' streak plates:
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We hope that in the future our plates will look like the ones on the left. We refer to the AddGene protocol here but note that their protocol here is for transformed bacteria, which we haven't made yet. This is actually a great reference site that we would like to promote here as well.
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Kirby-Baur Agar Diffusion Test-
Looking for Antimicrobial Compounds in Plants All Around Us!
We practiced this method using erythromycin and a few other compounds we thought might inhibit bacterial growth. The first time we had complete failures as no bacteria grew whatsoever! Only moulds on the discs due to contamination (we didn't sterilize the discs used). The second time we tried it we had much better success. Two students investigated the effects of ethanol extractions of several herbs on E. coli growth and found some interesting results. These images are taken from the CDC's website page on pathogenic bacteria- we do not use pathogenic bacteria in our school lab!
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Estimating Yeast Cell Density with Spectrophotometer
We used our LoggerPro Spectrovis Plus Spectrophotometer to measure cell density of bacterial and yeast cells in liquid media. The absorbance at 500 nm or Optical Density is directly tied to cell density. We plan on plating our dilution factors to determine numbers of bacteria per mL but are running out of petri dishes! We pan on using data for E. coli in our lab to standardize concentrations of bacteria in all our transformation labs.
Lab wish list: 500 petri dishes & vortex machine! |