Population Estimation Lab Report
methods of population estimation
November 18, 2014
BIOL 1121: General Biology II Lab
Fall 2014
Abstract Mark and recapture is a method commonly used in ecology to estimate an animal population 's size. A portion of the population is captured, marked, and released. This lab provides methods that can be used to estimate a
provided additional information for a better interpretation of lichen diversity values in biomonitoring studies of air pollution.
Introduction This section is an introduction to the lab objectives and its practical uses. It should normally be 1 to 2 paragraphs in a short report. The primary functions of an introduction are to describe the objectives of this laboratory exercise. The section needs to introduce the lab function and background. Any new concepts and terminology need to be given as well. Last should be the objectives of the lab. Include what should be accomplished or learned through performing this experiment.
For example, what is gel filtration chromatography? What does egg white consist of? What were you trying to getting out of the egg white sample? What properties of the technique did make it suitable for your purpose? What did you expect from the results? Why did you fractionate the protein? What did you do with the fractionated protein?
Materials and methods
Part I …show more content…
Two empty, sterile 1.5 ml microcentrifuge tubes and another one containing 600 µl of transformation solution (CaCl2) were obtained. One of the closed microcentrifuge was labeled +pGLO and the other –pGLO and then placed on a microcentrifuge tube rack. 250 µl of the transformation solution was then transferred to the tubes using a P-1000 micropipette with a sterile tip. The tubes were then placed in ice as the bacterial suspension was being prepared. 2 large (~2 mm in diameter) or 3 small colonies (<2 mm in diameter) of bacteria were picked from the starter plate using a sterile loop. The loop was then immersed in the transformation solution of the +pGLO and spun vigorously using the index finger and thumb, while the tube was held between the other index finger and thumb, until the entire colony was completely dispersed in the transformation solution. The +pGLO tube was then placed back in the tube rack on the ice. The same above steps were used for the –pGLO tube with the use of a new sterile loop. A vial containing rehydrated pGLO plasmids was obtained. 10 µl of the pGLO plasmid was then pipetted to the +pGLO tube using a fresh sterile tip. The tube was then closed amd returned to the foam rack on ice. No plasmid was added to the –pGLO tube. The tubes were then incubated in ice for 10 minutes. The tubes should stick out of the foam rack and have direct contact with ice. Four LB nutrient agar plates were then appropriately labeled:
a) LB plate: -pGLO
b) LB/amp plate: -pGLO
c) LB/amp plate: +pGLO
d) LB/amp/ara plate: +pGLO
The tubes while still in ice were brought and placed in a floating rack inside a 420C water bath and then incubated for 50 seconds. They were then immediately placed directly on ice without using the foam rack and incubated for 2 minutes. The tubes were then removed from the ice and placed on a microcentrifuge tube rack. Using a new sterile pipette tip, 250 µl of LB nutrient broth was added to each tube and then re-closed. The tubes were mixed by gently tapping them with fingers. They were then incubated for 15 minutes in a 370C water bath. Disposable L-shaped cell spreaders were then obtained. The closed tubes were then flicked to mix and resuspend the bacterial cells. Fresh sterile pipette tips for each tube were then used to transfer the appropriate cell suspension on the pre-labeled culture plates as follows:
a) 200 µl of –pGLO cells onto the LB plate
b) 200 µl of –pGLO cells onto the LB/Amp plate
c) 200 µl of +pGLO cells onto the LB/Amp plate
d) 200 µl of +pGLO cells onto the LB/Amp/ara plate
A new sterile L-shaped spreader was then used for each plate to spread suspensions evenly around the surface of the agar by the straight edge of the spreader being quickly skated on the entire plate surface. Contamination was prevented by not widely opening the lid while being inoculated. The plates were then left to sit for five minutes at room temperature to allow the liquid of the suspension to be soaked by the culture medium. Then they were stacked up and tied together with two #64 rubber bands. The plates were then labeled, placed upside down (inverted) and inoculated at 370C for 24-36 hours and then stored at 40C.
Part II
Observations were made on the bacterial growth on each plate to identify the transformed and un-transformed cells. A photograph of the plates on the UV transilluminator was taken. The observations of the petri dishes regarding bacterial growth, abundance of colonies and overall change in size were made under visible light. The color of the bacterial colonies were made under UV lamp and recorded in the Table 1 below.
Table 1: Number, Size and Color of colonies under visible and ultraviolet light
Plates
Growth
Patterna
Relative Number of colonies before and after transformationb
Colony Size before and after Transformationc
Color of colonies Under Visible lightd Color of Colonies under UV lightd
LB:-pGLO
LB/Amp:-pGLO
LB/Amp:+pGLO
LB/Amp/ara:+pGLO
a. Lawn of bacteria (uncountable) or separate round colonies (countable)
b. Numerous, many, some, or none
c. Larger, smaller or similar
d. Off-white or bright green
The number of colonies growing on each plate was counted and recorded on Table 2 below:
Table 2: Number of colonies counted under visible and ultraviolet light
Plates
# of colonies under visible light
# of colonies under UV light
LB: -pGLO
LB/Amp: -pGLO
LB/Amp: +pGLO
LB/Amp/ara: +pGLO
We determined the total number of successfully transformed cells from the result of the table. Then the amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate was determined in micrograms. The number of colonies on the LB/amp/ara plate was also calculated. From this data, we then finally calculated the efficiency of the pGLO transformation.
Results:
On the both left petri dishes, the plates were inoculated with an untransformed DNA (-pGLO) bacteria. The –pGLO LB formed a lawn of bacteria, while the -pGLO LB/amp had no sign of growth in it due to the presence of antibiotic (ampicillin). The plates on the right were inoculated with transformed bacteria which are resistant to ampicillin. The bright green fluorescent color on the +pGLO LB/amp/ara comes from the gene arabinose operon, (ara) responsible for lighting up by the ultraviolet light from the transilluminator.
Table 1: Number, Size and Color of colonies under visible and ultraviolet light
Plates
Growth
Patterna
Relative Number of colonies before and after transformationb
Colony Size before and after Transformationc
Color of colonies Under Visible lightd Color of Colonies under UV lightd
LB:-pGLO
uncountable numerous larger
Off white
Off white
LB/Amp:-pGLO
uncountable none clear clear LB/Amp:+pGLO countable many larger white white LB/Amp/ara:+pGLO countable many
yellow
Bright green
a. Lawn of bacteria (uncountable) or separate round colonies (countable)
b. Numerous, many, some, or none
c. Larger, smaller or similar
d. Off-white or bright green
Table 2: Number of colonies counted under visible and ultraviolet light
Plates
# of colonies under visible light
# of colonies under UV light
LB: -pGLO uncountable uncountable
LB/Amp: -pGLO none none
LB/Amp: +pGLO
300
300
LB/Amp/ara: +pGLO
270
270
The total amount of DNA was determined by: DNA (µg) = concentration of DNA (µg/µl) × Volume of DNA (µl) = 0.08 µg/µl × 10 µl = 0.8 µg
Fraction of DNA used= Volume spread on LB/amp/ara plate Total volume in the test tube = 200 µl 510 µl = 20 51
To calculate the number of micrograms of DNA spread on the LB/amp/ara it was calculated as follows: pGLO DNA spread (µg)= Total amount of DNA used (µg) × fraction of DNA pGLO DNA spread= 0.3137 µg
This is the number of DNA spread on the agar plate.
From the above calculations we determined:
Number of colonies on LB/amp/ara plate = 270
Micrograms of pGLO DNA spread on the plate = 0.3137 µg
Transformation efficiency = total number of cells growing on the agar plate Amount of DNA spread on the agar plate
= 860.69
discussion First, include a brief (consisting of 1 or 2 paragraph) conclusion. Refer to the objectives of the lab, and articulate whether or not they were met, and how. If some objectives were not met, give a brief explanation of why. Explain what was found or learned by performing this lab. Any ideas for improving the lab can also be included in this section.
Does the experiment support your hypothesis (when the experiment was to answer a biological question)?
Did the experiment showed expected results (when the experiment was not to answer the question)? For example, was the protein sample fractionated as expected and was the number of fractions you collected close to what you expected? If not, what could be the cause of the unexpected results? Describe the sources, or suspected sources of error. Describe all the problems in the procedure (how and why it did not work). What should you do in the next step? If the lab could be repeated, how should the procedure be changed to improve the
results?
Explain what was found and how it can be used in the future.
Whether the colorimetric spectrophotometry results were consistent with your simple color test you carried out when collecting fractions. Which fraction had the highest and the lowest concentration of protein? Which fraction had the highest and the lowest amount of total protein? Was a fraction highest in both concentration and amount of total protein? Explain why or why not.
References
Dept. Biol. Sci. 2012. General Biology Laboratory I Manual. Dept. Biol. Sci., University of Memphis, Memphis, TN.
---- Include others if you have additional references by following the format shown below. In general, the references are alphabetically ordered by the first author’s last name. ---
Avery, O.T., C.M. Macleod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of Pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J Exp Med. 79:137-158.
Biotechnology Explorer Team. 2011. Biotechnology ExplorerTM pGLOTM Bacterial Transformation Kit (Catalog No. 166-0003-EDU) Intruction Manual, Rev G. Bio-Rad Laboratories, Hercules, CA.
Chen, I., and D. Dubnau. 2004. DNA uptake during bacterial transformation. Nat Rev Microbiol. 2:241-249.
Griffith, F. 1928. The significance of pneumococcal types. J Hyg (Lond). 27:113-159.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 166:557-580.
Johnson, W.G., V.M. Davis, G.R. Kruger, and S.C. Weller. 2009. Influence of glyphosate-resistant cropping systems on weed species shifts and glyphosate-resistant weed populations. Eur J Agron. 31:162-172.
Nilsson, J., P. Jonasson, E. Samuelsson, S. Stahl, and M. Uhlen. 1996. Integrated production of human insulin and its C-peptide. Journal of biotechnology. 48:241-250.
Thim, L., M.T. Hansen, K. Norris, I. Hoegh, E. Boel, J. Forstrom, G. Ammerer, and N.P. Fiil. 1986. Secretion and processing of insulin precursors in yeast. Proceedings of the National Academy of Sciences of the United States of America. 83:6766-6770.
Thompson, G.A., W.R. Hiatt, D. Facciotti, D.M. Stalker, and L. Comai. 1987. Expression in plants of a bacterial gene coding for glyphosate resistance. Weed Sci. 35:19-23. van den Eede, G., H. Aarts, H.J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B. Jacobsen, T. Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, and A. Wilcks. 2004. The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 42:1127-1156.
Walmsley, A.M., and C.J. Arntzen. 2000. Plants for delivery of edible vaccines. Current opinion in biotechnology. 11:126-129.
Finishing Touches
1. Be sure you give your section number, group number, your name, your partner 's name and the date you did the activity.
2. Proofread one last time to be sure that you have used correct grammar and punctuation. Remember that Spell Check on the computer will not catch a word that is used incorrectly if it is spelled correctly (e.g., there-their).
3. You can use the grammar check to help you with the technical style of writing. Open the Tools menu and select Options. Click on the Spelling & Grammar tab and find the Writing style menu (near the bottom right corner). It is probably set on Standard. Open the drop-down menu and select Technical. Click OK. Open the Tools menu again and select Spelling and Grammar. The Grammar Checker will offer suggestions to formalize your writing in the technical style. When you are finished, reset the Writing Style to Standard.
4. Use serif (e.g., Times New Roman) or sans-serif (e.g. Arial) fonts, which are commonly used for scientific publication. Do not use fonts such as Comic Sans MS or Script. In publication, body text and section titles are usually typed in Times New Roman and the table title, table contents, and figure legends are typed in Arial or other sans-serif fonts.
5. Use Excel program to generate graphs and charts and “Insert” it. Do not “Link to file”. Hand-drawings are acceptable for diagrams and illustrations.
HOW TO USE THIS TEMPLATE
1. Save this Word file on your computer hard drive for your future use without changing the filename.
2. While maintaining the format, replace the highlighted text with your own contents removing the highlight. You may change the format as long as you maintain the section title in order. Do not make changes in the running footer section, which includes the file name, page number and current date.
November 18, 2014
BIOL 1121: General Biology II Lab
Fall 2014
Abstract Mark and recapture is a method commonly used in ecology to estimate an animal population 's size. A portion of the population is captured, marked, and released. This lab provides methods that can be used to estimate a
provided additional information for a better interpretation of lichen diversity values in biomonitoring studies of air pollution.
Introduction This section is an introduction to the lab objectives and its practical uses. It should normally be 1 to 2 paragraphs in a short report. The primary functions of an introduction are to describe the objectives of this laboratory exercise. The section needs to introduce the lab function and background. Any new concepts and terminology need to be given as well. Last should be the objectives of the lab. Include what should be accomplished or learned through performing this experiment.
For example, what is gel filtration chromatography? What does egg white consist of? What were you trying to getting out of the egg white sample? What properties of the technique did make it suitable for your purpose? What did you expect from the results? Why did you fractionate the protein? What did you do with the fractionated protein?
Materials and methods
Part I …show more content…
Two empty, sterile 1.5 ml microcentrifuge tubes and another one containing 600 µl of transformation solution (CaCl2) were obtained. One of the closed microcentrifuge was labeled +pGLO and the other –pGLO and then placed on a microcentrifuge tube rack. 250 µl of the transformation solution was then transferred to the tubes using a P-1000 micropipette with a sterile tip. The tubes were then placed in ice as the bacterial suspension was being prepared. 2 large (~2 mm in diameter) or 3 small colonies (<2 mm in diameter) of bacteria were picked from the starter plate using a sterile loop. The loop was then immersed in the transformation solution of the +pGLO and spun vigorously using the index finger and thumb, while the tube was held between the other index finger and thumb, until the entire colony was completely dispersed in the transformation solution. The +pGLO tube was then placed back in the tube rack on the ice. The same above steps were used for the –pGLO tube with the use of a new sterile loop. A vial containing rehydrated pGLO plasmids was obtained. 10 µl of the pGLO plasmid was then pipetted to the +pGLO tube using a fresh sterile tip. The tube was then closed amd returned to the foam rack on ice. No plasmid was added to the –pGLO tube. The tubes were then incubated in ice for 10 minutes. The tubes should stick out of the foam rack and have direct contact with ice. Four LB nutrient agar plates were then appropriately labeled:
a) LB plate: -pGLO
b) LB/amp plate: -pGLO
c) LB/amp plate: +pGLO
d) LB/amp/ara plate: +pGLO
The tubes while still in ice were brought and placed in a floating rack inside a 420C water bath and then incubated for 50 seconds. They were then immediately placed directly on ice without using the foam rack and incubated for 2 minutes. The tubes were then removed from the ice and placed on a microcentrifuge tube rack. Using a new sterile pipette tip, 250 µl of LB nutrient broth was added to each tube and then re-closed. The tubes were mixed by gently tapping them with fingers. They were then incubated for 15 minutes in a 370C water bath. Disposable L-shaped cell spreaders were then obtained. The closed tubes were then flicked to mix and resuspend the bacterial cells. Fresh sterile pipette tips for each tube were then used to transfer the appropriate cell suspension on the pre-labeled culture plates as follows:
a) 200 µl of –pGLO cells onto the LB plate
b) 200 µl of –pGLO cells onto the LB/Amp plate
c) 200 µl of +pGLO cells onto the LB/Amp plate
d) 200 µl of +pGLO cells onto the LB/Amp/ara plate
A new sterile L-shaped spreader was then used for each plate to spread suspensions evenly around the surface of the agar by the straight edge of the spreader being quickly skated on the entire plate surface. Contamination was prevented by not widely opening the lid while being inoculated. The plates were then left to sit for five minutes at room temperature to allow the liquid of the suspension to be soaked by the culture medium. Then they were stacked up and tied together with two #64 rubber bands. The plates were then labeled, placed upside down (inverted) and inoculated at 370C for 24-36 hours and then stored at 40C.
Part II
Observations were made on the bacterial growth on each plate to identify the transformed and un-transformed cells. A photograph of the plates on the UV transilluminator was taken. The observations of the petri dishes regarding bacterial growth, abundance of colonies and overall change in size were made under visible light. The color of the bacterial colonies were made under UV lamp and recorded in the Table 1 below.
Table 1: Number, Size and Color of colonies under visible and ultraviolet light
Plates
Growth
Patterna
Relative Number of colonies before and after transformationb
Colony Size before and after Transformationc
Color of colonies Under Visible lightd Color of Colonies under UV lightd
LB:-pGLO
LB/Amp:-pGLO
LB/Amp:+pGLO
LB/Amp/ara:+pGLO
a. Lawn of bacteria (uncountable) or separate round colonies (countable)
b. Numerous, many, some, or none
c. Larger, smaller or similar
d. Off-white or bright green
The number of colonies growing on each plate was counted and recorded on Table 2 below:
Table 2: Number of colonies counted under visible and ultraviolet light
Plates
# of colonies under visible light
# of colonies under UV light
LB: -pGLO
LB/Amp: -pGLO
LB/Amp: +pGLO
LB/Amp/ara: +pGLO
We determined the total number of successfully transformed cells from the result of the table. Then the amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate was determined in micrograms. The number of colonies on the LB/amp/ara plate was also calculated. From this data, we then finally calculated the efficiency of the pGLO transformation.
Results:
On the both left petri dishes, the plates were inoculated with an untransformed DNA (-pGLO) bacteria. The –pGLO LB formed a lawn of bacteria, while the -pGLO LB/amp had no sign of growth in it due to the presence of antibiotic (ampicillin). The plates on the right were inoculated with transformed bacteria which are resistant to ampicillin. The bright green fluorescent color on the +pGLO LB/amp/ara comes from the gene arabinose operon, (ara) responsible for lighting up by the ultraviolet light from the transilluminator.
Table 1: Number, Size and Color of colonies under visible and ultraviolet light
Plates
Growth
Patterna
Relative Number of colonies before and after transformationb
Colony Size before and after Transformationc
Color of colonies Under Visible lightd Color of Colonies under UV lightd
LB:-pGLO
uncountable numerous larger
Off white
Off white
LB/Amp:-pGLO
uncountable none clear clear LB/Amp:+pGLO countable many larger white white LB/Amp/ara:+pGLO countable many
yellow
Bright green
a. Lawn of bacteria (uncountable) or separate round colonies (countable)
b. Numerous, many, some, or none
c. Larger, smaller or similar
d. Off-white or bright green
Table 2: Number of colonies counted under visible and ultraviolet light
Plates
# of colonies under visible light
# of colonies under UV light
LB: -pGLO uncountable uncountable
LB/Amp: -pGLO none none
LB/Amp: +pGLO
300
300
LB/Amp/ara: +pGLO
270
270
The total amount of DNA was determined by: DNA (µg) = concentration of DNA (µg/µl) × Volume of DNA (µl) = 0.08 µg/µl × 10 µl = 0.8 µg
Fraction of DNA used= Volume spread on LB/amp/ara plate Total volume in the test tube = 200 µl 510 µl = 20 51
To calculate the number of micrograms of DNA spread on the LB/amp/ara it was calculated as follows: pGLO DNA spread (µg)= Total amount of DNA used (µg) × fraction of DNA pGLO DNA spread= 0.3137 µg
This is the number of DNA spread on the agar plate.
From the above calculations we determined:
Number of colonies on LB/amp/ara plate = 270
Micrograms of pGLO DNA spread on the plate = 0.3137 µg
Transformation efficiency = total number of cells growing on the agar plate Amount of DNA spread on the agar plate
= 860.69
discussion First, include a brief (consisting of 1 or 2 paragraph) conclusion. Refer to the objectives of the lab, and articulate whether or not they were met, and how. If some objectives were not met, give a brief explanation of why. Explain what was found or learned by performing this lab. Any ideas for improving the lab can also be included in this section.
Does the experiment support your hypothesis (when the experiment was to answer a biological question)?
Did the experiment showed expected results (when the experiment was not to answer the question)? For example, was the protein sample fractionated as expected and was the number of fractions you collected close to what you expected? If not, what could be the cause of the unexpected results? Describe the sources, or suspected sources of error. Describe all the problems in the procedure (how and why it did not work). What should you do in the next step? If the lab could be repeated, how should the procedure be changed to improve the
results?
Explain what was found and how it can be used in the future.
Whether the colorimetric spectrophotometry results were consistent with your simple color test you carried out when collecting fractions. Which fraction had the highest and the lowest concentration of protein? Which fraction had the highest and the lowest amount of total protein? Was a fraction highest in both concentration and amount of total protein? Explain why or why not.
References
Dept. Biol. Sci. 2012. General Biology Laboratory I Manual. Dept. Biol. Sci., University of Memphis, Memphis, TN.
---- Include others if you have additional references by following the format shown below. In general, the references are alphabetically ordered by the first author’s last name. ---
Avery, O.T., C.M. Macleod, and M. McCarty. 1944. Studies on the chemical nature of the substance inducing transformation of Pneumococcal types: Induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J Exp Med. 79:137-158.
Biotechnology Explorer Team. 2011. Biotechnology ExplorerTM pGLOTM Bacterial Transformation Kit (Catalog No. 166-0003-EDU) Intruction Manual, Rev G. Bio-Rad Laboratories, Hercules, CA.
Chen, I., and D. Dubnau. 2004. DNA uptake during bacterial transformation. Nat Rev Microbiol. 2:241-249.
Griffith, F. 1928. The significance of pneumococcal types. J Hyg (Lond). 27:113-159.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 166:557-580.
Johnson, W.G., V.M. Davis, G.R. Kruger, and S.C. Weller. 2009. Influence of glyphosate-resistant cropping systems on weed species shifts and glyphosate-resistant weed populations. Eur J Agron. 31:162-172.
Nilsson, J., P. Jonasson, E. Samuelsson, S. Stahl, and M. Uhlen. 1996. Integrated production of human insulin and its C-peptide. Journal of biotechnology. 48:241-250.
Thim, L., M.T. Hansen, K. Norris, I. Hoegh, E. Boel, J. Forstrom, G. Ammerer, and N.P. Fiil. 1986. Secretion and processing of insulin precursors in yeast. Proceedings of the National Academy of Sciences of the United States of America. 83:6766-6770.
Thompson, G.A., W.R. Hiatt, D. Facciotti, D.M. Stalker, and L. Comai. 1987. Expression in plants of a bacterial gene coding for glyphosate resistance. Weed Sci. 35:19-23. van den Eede, G., H. Aarts, H.J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B. Jacobsen, T. Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, and A. Wilcks. 2004. The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association. 42:1127-1156.
Walmsley, A.M., and C.J. Arntzen. 2000. Plants for delivery of edible vaccines. Current opinion in biotechnology. 11:126-129.
Finishing Touches
1. Be sure you give your section number, group number, your name, your partner 's name and the date you did the activity.
2. Proofread one last time to be sure that you have used correct grammar and punctuation. Remember that Spell Check on the computer will not catch a word that is used incorrectly if it is spelled correctly (e.g., there-their).
3. You can use the grammar check to help you with the technical style of writing. Open the Tools menu and select Options. Click on the Spelling & Grammar tab and find the Writing style menu (near the bottom right corner). It is probably set on Standard. Open the drop-down menu and select Technical. Click OK. Open the Tools menu again and select Spelling and Grammar. The Grammar Checker will offer suggestions to formalize your writing in the technical style. When you are finished, reset the Writing Style to Standard.
4. Use serif (e.g., Times New Roman) or sans-serif (e.g. Arial) fonts, which are commonly used for scientific publication. Do not use fonts such as Comic Sans MS or Script. In publication, body text and section titles are usually typed in Times New Roman and the table title, table contents, and figure legends are typed in Arial or other sans-serif fonts.
5. Use Excel program to generate graphs and charts and “Insert” it. Do not “Link to file”. Hand-drawings are acceptable for diagrams and illustrations.
HOW TO USE THIS TEMPLATE
1. Save this Word file on your computer hard drive for your future use without changing the filename.
2. While maintaining the format, replace the highlighted text with your own contents removing the highlight. You may change the format as long as you maintain the section title in order. Do not make changes in the running footer section, which includes the file name, page number and current date.