Team:RHIT/Protocol.html
From 2013.igem.org
- Protocols
- Growing Cells
- Mini Prep
- DNA Purification
- Electrophoresis Gel
- DNA Recovery
- DNA Digest
- Ligation
- Transformation
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In lab we used a variety of protocols that helped us achieve our goal of constructing a platform for interspecies dependence.
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COMPONENT | 20 μl REACTION |
---|---|
10X T4 DNA Ligase Buffer* | 2 μl |
Vector DNA (3 kb) | 50 ng (0.025 pmol) |
Insert DNA (1 kb) | 50 ng (0.076 pmol) |
Nuclease-free water | to 20 μl |
T4 DNA Ligase | 1 μl |
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The systems (one for E. coli and one for Yeast) described during the project explanation are broken up into their parts below. Each of the parts combined create a system that has a specific function but the two constructs together create a positive feedback loop that allows the two organisms to stay in close proximity.
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E. coli and yeast strains are being grown on plates. Cells needs specific nutrients in order to grow and produce more DNA. The Jello-like material (agar) that is spread in an even layer on the plastic plate allows for the growth of cells. The cells are applied to the agar. Each type of cell needs to grow on a separate plate to ensure that we know what’s on the plate. The plates are placed in a warm area where they can grow the quickest. The plates need to sit in the warmth at least overnight in order to see any cells. The cells, in our case, will be round and tan in color.
Inoculation of a Liquid Culture:Once the cells are grown on the agar, they can be picked off one at a time. The cell that is picked off of the plate is scraped off into a tube filled with liquid nutrients. This liquid is very similar to the agar in nutrient content.
The liquid containing the cell is then again growth in warm place overnight. The liquid should change from being very clear (able to be seen through) to being very cloudy. This cloudy material is growing cells
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The point of a mini prep is to obtain and clean the DNA that is within these cells that we just grew up in the liquid nutrients. The process begins by placing some of the cloudy solution into a small tube and using a machine to spin the cells that are in the liquid down to the bottom of the tube. These cells form a clump at the bottom of the tube and the liquid that does not contain cells stays floating above the clump of cells. The liquid is dumped down the sink because it does not contain cells with DNA. The clump of cells is kept in the small tube.
Next, a detergent is added to the tube with the cells. The detergent is kind of like a soap that breaks the membrane layer surrounding the cell open. The layer has to be broken in order to reach the DNA that is contained inside of it. The tube is then spun again and another clump is formed at the bottom of the tube. This time the clump is made up of the broken parts of the cell and the liquid floating above it has the DNA in it. The liquid is kept and the clump is thrown away.
This liquid DNA is poured onto a filter that grabs the DNA particles and holds onto them. The filter that has the DNA attached to it is then washed a few times with salty solutions to clean the DNA. Then, the DNA is washed off of the filter (using extra clean water) into a clean tube. The DNA can be used for another experiment or can be stored. DNA must be stored in a freezer to keep it from falling apart.
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DNA purification is a process used to take many samples of DNA (samples that most likely were obtained from the Mini Prep process) and combine then into a single concentrated solution of DNA. The multiple samples are first all dumped into the same tube. A buffer solution (salty liquid) is added to the tube and then ethanol is added to convert the originally liquid DNA solution into solid DNA material. This is called precipitating the DNA. Now that the DNA is separated from the other liquids a machine called a centrifuge is used to spin the tube very quickly, forcing the heavy, solid DNA material to the bottom of the tube. The liquid that was in the tube separates from the solid DNA by floating on top of the solid. This liquid does not contain DNA and is therefore removed from the tube. The solid DNA that is located at the bottom of the tube is then dissolved in a small volume of water. The volume of the sample decreases significantly, but still contains all of the DNA that was there before therefore increasing the concentration of the solution.
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DNA is like a fingerprint, meaning that each DNA sequence is unique. These fingerprints are used to identify the different pieces of DNA by size without actually knowing the sequence. The size of DNA can be very useful when trying to distinguish between different pieces of DNA quickly.
The way that scientists find the size of DNA is by using gel electrophoresis. The negative charge of DNA allows for movement of DNA particles to be controlled and the movement measured. This big word, electrophoresis, just means that the DNA particles are moving because of electricity. Mechanically, the DNA is loaded into holes that are made in an agarose gel. The process involves the movement of DNA particles through a medium that is called agarose. The agarose is a jelly-like material that serves as a grid for DNA to move on. Once the DNA is placed in the holes, electric current is applied to the agarose gel. The electric current causes the negative DNA particles to move toward the positive end of the current. As the DNA moves down the agarose gel, separation of different bands occurs. The separation is based on the size of the DNA. It takes a few hours for full separation of bands to happen. After the separation has occurred, the bands of different DNA sizes can be seen by shining UV light on the agarose gel.
As stated above, each band represents DNA of a different size. The bigger the piece of DNA, the slower it will move; while if the DNA piece is small it will move down the agarose gel quickly.
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Once DNA is run on an agarose gel, the DNA can be used again by cutting it out of the agarose and cleaning the portion of the agarose gel cut out so that only the DNA is obtained. This is an excellent method when multiple pieces of DNA are in one sample, the scientist knows the size of the DNA that they want to obtain, and they want only one of the pieces of DNA by itself.
If DNA is observed as a band on the agarose gel, the band of interest is cut using a knife from the gel. This piece of agarose containing the DNA is placed in a tube and is placed in hot water to allow the agarose to melt. Once the agarose is melted, the sample of DNA is then cleaned very well to insure that only DNA now exists in the sample.
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When you think of the name of this process “digest,” think of your stomach and what it means for your stomach to digest food. Usually digest means to break down. This is the same concept as a digest performed on a sample of DNA. In the case of digesting DNA, what actually works to break it down is what is called a restriction enzyme. These restriction enzymes, when put in a certain environment, can cut the DNA at specific sites. In some cases the enzymes can even cut the DNA at more than one site. The reason that scientists would want to cut/break down pieces of DNA is to obtain only the part of the DNA that the scientist is wishing to use. This part of DNA is usually of importance due to a special function that it might have.
The process begins with DNA in a tube. Added to the DNA is the restriction enzyme and a solution called a buffer that provides the best environment for the enzyme to cut out. It takes a few hours for the enzymes to cut the DNA. The different pieces of DNA that result from the digestion can be observed using the gel electrophoresis procedure explained above.
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Ligate means to bind. Ligation is just that: it involves the binding of multiple pieces of DNA. These pieces are created by using restriction enzymes. The reason why a scientist would want to bind multiple pieces of DNA is in order to combine the function of multiple pieces of DNA into one segment of DNA. The segment of DNA created from ligation is called a plasmid. A plasmid is a circular piece of DNA.
To prepare the DNA for ligation, the DNA in the tubes is first heated for about 30 minutes. The heat is supposed to kill the restriction enzymes that may still be present in the DNA sample. A portion of the two samples of DNA are added to a new tube along with ligase. Ligase helps in the binding of these two segments. This tube is incubated for about ten minutes and the process is done. The success of the ligation can be assessed by using gel electrophoresis. What is expected to be seen is one large band that is the sum of the two smaller pieces of DNA.
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In order to perform this process, the DNA used must circular (i.e. a plasmid must be used). Usually after a ligation is performed, a transformation is what follows. The idea of a transformation is to combine the ligated DNA with DNA for what is called a competent cell. Competent cells are cells that take up and express DNA readily. The ligated DNA is put with the cells and the cells are put on an agar plate. The idea is that the ligated DNA will be taken up and expressed by the competent cells that grow on the plate. A liquid culture can then be grown using these cells. Then, the DNA can be purified using the Mini Prep procedure.