Lab 6: Isolation Of Chromosomal DNA
Lab 6: Isolation of Chromosomal DNA
Mic 428L/ Section 001
Introduction: In biological research to address and eventually answer a multitude of questions, usually involves isolating chromosomal DNA. The purpose in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward, the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the Tn.
The isolation of genomic …show more content…
DNA is a prerequisite for many types of experiments including DNA sequencing and cloning. One common goal of the isolation of the chromosomal DNA is to avoid shearing. The large chromosome is fragile and can be sheared very easily. Although there will always be some chromosomal DNA that cannot avoid shearing, most methods use a gentle lysis step using a detergent, SDS, and a proteinase enzyme to degrade proteins. Chloroform will also be used in the isolation procedure to remove any additional proteins bound to the DNA that we do not want. Additionally, the chromosomal DNA will precipitate out using a non polar solvent, isopropanol or ethanol. DNA is polar because of the negative charge on its phosphate backbone. Therefore, when it is exposed to a less polar solvent, such as ethanol or isopropanol, it will reduce the amount of surface area that is exposed to that solvent therefore it will form a ball and precipitate out.
The chromosomal DNA will be digested with a restriction enzyme once it has been isolated. We will use the restriction enzyme BamHI which cuts in fragments of around 5,000 base pairs. This means that BamHI will digest the chromosome once every 5,000 base pairs. For a genome of 6 Mb, BamHI will digest it into 1,200 fragments. One of our goals of this experiment was to self ligate these fragments. The idea behind this is that only one of these self ligated fragments will contain the Tn, therefore, if we transform all of them into E. Coli, only the circular piece containing the Tn with the π dependent R6 origin of replication will replicate.
Many bacteria in nature are able to naturally take up exogenous DNA from the environment. E. Coli, the bacteria that we are working with, however, does not, and needs to made competent in order to take up the exogenous DNA by electroporation. In this procedure, E. Coli is made competent by adding CaCl2, and glycerol. To transform the DNA into the competent E.Coli, electroporation is used. Electroporation is an increase in electrical conductivity and membrane permeability as a result of an applied electric field.
Methods:
Purification of Genomic DNA: An overnight culture of the mutants selected is prepared and transferred to a 1.5 ml microfuge tube on ice. The cells are pelleted by centrifugation. The supernatant is discarded and Cell Lysis solution is added and mixed. Cell lysis solution is a detergent that is used to lyse the cells and avoids shearing the DNA. RNase A solution is then added to remove any additional proteins. Protein precipitation solution is then added to allow for the chromosomal DNA to precipitate out. Isopropanol is then added to the previous supernatant. Isopropanol is a non polar solvent. DNA is very polar therefore when exposed to a polar solvent it will reduce its surface area exposed to the solvent and will collapse into a ball. The DNA should be visible as a small, white, pellet. Ethanol is then added to wash the DNA pellet of any remaining contaminants. The supernatant is then discarded, and the tube is turned upside down, making sure the pellet remains inside. Hydration solution is then added and the mix is vortex in order to break apart the pellet. The sample is then incubated at room temperature overnight with gently shaking.
Chromosomal Digestion
Our isolated chromosomal DNA from the previous step with them be mixed in with BamHI in order to digest it into many small fragments. They are then incubated and allowed to digest in a water bath for 2 hours.
Ligations
The restricted digested chromosomal DNA will then be mixed with ligase solution so that DNA ligase can self ligate the fragments into circles. A ligase buffer and water is also added to this solution. They are then incubated overnight and stored until the next lab period.
Transformation by Electroporation
2μl of the ligation mix will be added to a 1.5ml chilled tube of electrocompetent EC1000 pir-116, which is the strain of E.coli that has already been made competent for us.
22μl will then be added to a chilled electroporation cuvette. The cells will be electroporated using a Bio-Rad Gene Pulser, which will send electrical pulses through the E.coli so that the exogenous DNA can enter it. Room temperature SOC buffer is then added to stabilize the cells after being in a state of stress from the electroporation. The solution is then transferred to a new 1.5 ml microfuge tube and stored at 37°C for 60 minutes. 200μl of the solution will then be spread on an LB+Kan50 plate to select for the plasmid that contains the kanamycin resistance marker. These plates will be incubated at 37°C for 1-3 days. After 1-3 days, we will select 2 colonies from each plate and retouch a new LB+Kan50plate in order to ensure that we obtained the plasmid DNA.
Isolation of plasmid DNA for sizing and sequencing
The isolated plasmid DNA will then be run on an agarose gel to determine the size of it. Tracking dye will be added to the solution which contains glycerol that will weigh the samples down in the wells.
Results:
Kb ladder
Well 3
Well 7
Well 10
Figure 1: Figure 1 shows our fragment that was run through gel electrophoresis. Our results are seen here in wells 3. The neighboring team used wells 7 and 10 with similar results.. The Kb ladder can be seen in well 1. The streaked appearance of the DNA is explained below.
Discussion: The results from the gel electrophoresis revealed our mutant fragment samples displayed a streaking pattern suggest that the chromosomes were interrupted during digestion procedure and therefore not cleaved properly. This in turn made ligation impossible, resulting in much too large DNA fragments for a reliable base pairs estimate. Had the experiment been successful, the length of the first mutant (well 3) should have been approximately 6,108 base pairs in length and the second mutant in wells 7 and 10 were believed to have equal lengths of 12,216 base pairs. These lengths were determined by using another group’s gel in conjunction with our own and using similarities between the two and comparing both to the Kb ladder in the first well of the gel. The results suggested that the gene that was interrupted in E. Coli strain DH5 αλpir for the first mutant was gltD, and for the second mutant was Bamb. The gene gltD encodes for the protein glutamate synthase which is responsible for giving the cell the ability to grow on many alternative nitrogen sources. Bacteria derive most of their nitrogen from either glutamate or glutamine. In E.coli specifically, about 88% of the cellular nitrogen in the cell is derived from glutamate. Interestingly, the preferred nitrogen source of enteric bacteria is ammonia. In bacteria, ammonia can be fixed into the carbon skeleton to form glutamate. Mutants that cannot accumulate glutamate enter into a nitrogen starvation response. The phenotype that we observed for this mutant was no growth on the M9 plate. The M9 plate is a minimal nutrient plate therefore ammonia or a compound associated with ammonia was probably very scarce and the gltD would have given the cell the ability to grow on alternative nitrogen sources such as glutamate. This gene was interrupted when the plasmid was inserted, therefore, there was no growth on the M9 plate because the gltD gene was interrupted.
Mic 428L/ Section 001
Introduction: In biological research to address and eventually answer a multitude of questions, usually involves isolating chromosomal DNA. The purpose in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward, the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the Tn.
The isolation of genomic …show more content…
DNA is a prerequisite for many types of experiments including DNA sequencing and cloning. One common goal of the isolation of the chromosomal DNA is to avoid shearing. The large chromosome is fragile and can be sheared very easily. Although there will always be some chromosomal DNA that cannot avoid shearing, most methods use a gentle lysis step using a detergent, SDS, and a proteinase enzyme to degrade proteins. Chloroform will also be used in the isolation procedure to remove any additional proteins bound to the DNA that we do not want. Additionally, the chromosomal DNA will precipitate out using a non polar solvent, isopropanol or ethanol. DNA is polar because of the negative charge on its phosphate backbone. Therefore, when it is exposed to a less polar solvent, such as ethanol or isopropanol, it will reduce the amount of surface area that is exposed to that solvent therefore it will form a ball and precipitate out.
The chromosomal DNA will be digested with a restriction enzyme once it has been isolated. We will use the restriction enzyme BamHI which cuts in fragments of around 5,000 base pairs. This means that BamHI will digest the chromosome once every 5,000 base pairs. For a genome of 6 Mb, BamHI will digest it into 1,200 fragments. One of our goals of this experiment was to self ligate these fragments. The idea behind this is that only one of these self ligated fragments will contain the Tn, therefore, if we transform all of them into E. Coli, only the circular piece containing the Tn with the π dependent R6 origin of replication will replicate.
Many bacteria in nature are able to naturally take up exogenous DNA from the environment. E. Coli, the bacteria that we are working with, however, does not, and needs to made competent in order to take up the exogenous DNA by electroporation. In this procedure, E. Coli is made competent by adding CaCl2, and glycerol. To transform the DNA into the competent E.Coli, electroporation is used. Electroporation is an increase in electrical conductivity and membrane permeability as a result of an applied electric field.
Methods:
Purification of Genomic DNA: An overnight culture of the mutants selected is prepared and transferred to a 1.5 ml microfuge tube on ice. The cells are pelleted by centrifugation. The supernatant is discarded and Cell Lysis solution is added and mixed. Cell lysis solution is a detergent that is used to lyse the cells and avoids shearing the DNA. RNase A solution is then added to remove any additional proteins. Protein precipitation solution is then added to allow for the chromosomal DNA to precipitate out. Isopropanol is then added to the previous supernatant. Isopropanol is a non polar solvent. DNA is very polar therefore when exposed to a polar solvent it will reduce its surface area exposed to the solvent and will collapse into a ball. The DNA should be visible as a small, white, pellet. Ethanol is then added to wash the DNA pellet of any remaining contaminants. The supernatant is then discarded, and the tube is turned upside down, making sure the pellet remains inside. Hydration solution is then added and the mix is vortex in order to break apart the pellet. The sample is then incubated at room temperature overnight with gently shaking.
Chromosomal Digestion
Our isolated chromosomal DNA from the previous step with them be mixed in with BamHI in order to digest it into many small fragments. They are then incubated and allowed to digest in a water bath for 2 hours.
Ligations
The restricted digested chromosomal DNA will then be mixed with ligase solution so that DNA ligase can self ligate the fragments into circles. A ligase buffer and water is also added to this solution. They are then incubated overnight and stored until the next lab period.
Transformation by Electroporation
2μl of the ligation mix will be added to a 1.5ml chilled tube of electrocompetent EC1000 pir-116, which is the strain of E.coli that has already been made competent for us.
22μl will then be added to a chilled electroporation cuvette. The cells will be electroporated using a Bio-Rad Gene Pulser, which will send electrical pulses through the E.coli so that the exogenous DNA can enter it. Room temperature SOC buffer is then added to stabilize the cells after being in a state of stress from the electroporation. The solution is then transferred to a new 1.5 ml microfuge tube and stored at 37°C for 60 minutes. 200μl of the solution will then be spread on an LB+Kan50 plate to select for the plasmid that contains the kanamycin resistance marker. These plates will be incubated at 37°C for 1-3 days. After 1-3 days, we will select 2 colonies from each plate and retouch a new LB+Kan50plate in order to ensure that we obtained the plasmid DNA.
Isolation of plasmid DNA for sizing and sequencing
The isolated plasmid DNA will then be run on an agarose gel to determine the size of it. Tracking dye will be added to the solution which contains glycerol that will weigh the samples down in the wells.
Results:
Kb ladder
Well 3
Well 7
Well 10
Figure 1: Figure 1 shows our fragment that was run through gel electrophoresis. Our results are seen here in wells 3. The neighboring team used wells 7 and 10 with similar results.. The Kb ladder can be seen in well 1. The streaked appearance of the DNA is explained below.
Discussion: The results from the gel electrophoresis revealed our mutant fragment samples displayed a streaking pattern suggest that the chromosomes were interrupted during digestion procedure and therefore not cleaved properly. This in turn made ligation impossible, resulting in much too large DNA fragments for a reliable base pairs estimate. Had the experiment been successful, the length of the first mutant (well 3) should have been approximately 6,108 base pairs in length and the second mutant in wells 7 and 10 were believed to have equal lengths of 12,216 base pairs. These lengths were determined by using another group’s gel in conjunction with our own and using similarities between the two and comparing both to the Kb ladder in the first well of the gel. The results suggested that the gene that was interrupted in E. Coli strain DH5 αλpir for the first mutant was gltD, and for the second mutant was Bamb. The gene gltD encodes for the protein glutamate synthase which is responsible for giving the cell the ability to grow on many alternative nitrogen sources. Bacteria derive most of their nitrogen from either glutamate or glutamine. In E.coli specifically, about 88% of the cellular nitrogen in the cell is derived from glutamate. Interestingly, the preferred nitrogen source of enteric bacteria is ammonia. In bacteria, ammonia can be fixed into the carbon skeleton to form glutamate. Mutants that cannot accumulate glutamate enter into a nitrogen starvation response. The phenotype that we observed for this mutant was no growth on the M9 plate. The M9 plate is a minimal nutrient plate therefore ammonia or a compound associated with ammonia was probably very scarce and the gltD would have given the cell the ability to grow on alternative nitrogen sources such as glutamate. This gene was interrupted when the plasmid was inserted, therefore, there was no growth on the M9 plate because the gltD gene was interrupted.