BISC 219: Assignment Help- Materials and Methods
Materials and Methods: Plasmid Isolation and Transformation
Students commonly have trouble with the methods section, primarily because it is mistakenly considered to be the place to describe exactly what you did in lab, step by step. Instead, think of this section as the place to present the progress of the experiment through a detailed but brief description of the methods used to collect and to analyze your data. Present enough information so that the reader can evaluate what you did, see how you achieved your experimental goals, and could, possibly, replicate your work. Do not clutter your paper with trivial or nonessential information. This section should not be a transcription of the lab manual or a chronological description of your lab experience. It should be as succinct as possible.
Divide the Methods section into subheadings with titles that give the goal as well as the main tool used to achieve the goal of each part of the experiment, e.g, “DNA fragmentation and separation by restriction enzyme digestion and agarose gel electrophoresis” is a better subheading than “Gel Electrophoresis” or “Identifying the Gene”.
Methods are always presented in the past tense because the experiments are complete. Third person is preferred, even if it requires passive voice, because avoiding first person helps you focus on the progress of the experiment rather than on your lab day (“DNA was extracted in 70% ethanol” is preferred over, “We added 1ml of 95% ethanol to a tube containing…”).
Never use the word “tube” (or similar words such as “plates”). Instead of stressing the container, explain what’s in it, using specific terms that increase understanding. You reader doesn’t want to know all about your lab day, but she is struggling to understand how you reached your goal from the starting materials. Rather than giving a recipe (e.g. mixed this vol. of this with this vol. of that), give effective concentrations of reagents when you describe crucial points in the experiment.
Example: “Product formation after 10 minutes was halted with the addition of 0.1M Na2CO3 to each enzyme/substrate reaction”, is preferred over “1ml of 1M Na2CO3 was added to each tube in 30 sec. intervals.”
How to calculate effective concentration: You should include ingredients and concentrations of all reagents at first mention, if you can. The ingredients and concentration of all stock solutions, suspensions, and reagents (or where to buy proprietary reagents) are found in the lab protocols or in a separate appendix. Give reagent “recipes” or supplier information in parenthesis after you first mention anything that will have to be made or bought. Many reagents are proprietary; therefore, showing your reader where to buy them is the best you can do if you can’t give enough information for your reader to be able to make them. Examples: Cells were cultured at 37C overnight in Luria broth (1% tyrptone, 0.5% yeast extract,1% NaCl ). DNA was extracted from leaf disks using a Hot Start® DNA extraction kit available from Invitrogen, Inc.
To avoid ambiguity, most scientific papers provide the effective concentration of reagents at the time of the reaction rather than giving volumes of various stock solutions as they are added together. Remember that you do not usually have to provide the effective concentrations of diluents.
Examples: 1. The effective concentration of ampicillin in broth during cell growth is calculated by multiplying the stock concentration (100 mg/ml) by the dilution created when 10 µl of the stock ampicillin was added to 10 ml (10,000 µl) of broth. In this case, the effective concentration of ampicillin in the culture is 0.1 mg/ml AMP because there is 10 µl of stock ampicillin in 10,000 µl (10 ml). This constitutes a 1/1000 dilution of the 100 mg/ml stock. Note that the concentrations of the components of the broth are not significantly changed by the addition of this small amount (10 µl) of ampicillin to 10,000 µl of LB, so the concentrations of the ingredients used to make the broth can be given as stock concentrations without adjustment.
2. If 100 µl of a solution containing 50 mM glucose, 10 mM EDTA and 25 mM Tris buffer was used to resuspend a bacterial cell pellet, the concentrations in the stock solution are not altered because the pelleted cells do not represent a measurable fluid volume.
3. When 200 µl of a stock solution, containing 0.2 M NaOH and 1% SDS, are added to 100 µl of suspended cells, the total volume is now 300 µl. The diluted solution contains effective concentrations of 0.132 M NaOH and 0.66% SDS. These effective concentrations were calculated by multiplying the stock concentrations by the fraction of the volume of the NaOH/SDS solution to the total reaction volume: 200 µl/300 µl = 2/3 or 0.66.
Sample partial Methods section: Restriction Enzyme Digestion and separation of plasmid fragments by electrophoresis Plasmid DNA was digested with a restriction enzyme by combining 20% plasmid pHM64 (Table1) and 20% plasmid p280-35 (Table 1) with 0.4% HindIII ( SigmalAdrich www.sigmaaldrich.com) for 20 min at 37C in High Salt Buffer (0.1M NaCl, 50 mM Tris pH 7.5, 10mM MgCl2, and 1mM DTT). The DNA fragments from the digestion were separated by agarose gel electrophoresis by applying 20µl of the digested DNA to a 1.5% agarose gel in TGE (0.25M Tris, 1.9M Glycine, 13mM EDTA) with 0.5% SYBR safe DNA stain from Invitrogen www.invitrogen.com. DNA fragments were subjected to electrophoresis at 100V for one hour. Stained and separated DNA fragments were visualized and photographed under UV light.
If you use mathematical formulas or statistical analyses, include an explanation of those calculations or statistical tools. Explain the variables that were compared, the types of statistical tests used, and for what they were used. If you calculate a p value, include the -level (threshold significance probability—often 0.05). Explain how measurements (original data collected) are transformed, perhaps by taking their logarithms, multiplying by a dilution factor, using a molar extinction co-efficient to transform Absorbance to concentration, etc.
Example of how you might write about a statistical analysis tool or a mathematical transformation from Absorbance to enzyme activity in your Methods section: Statistical Analysis: Averages and standard deviations of both the five and ten week counts were calculated and graphed as a column graph. A one-way ANOVA and Tukey HSD test were performed on both counts to compare the yeast/whole wheat combinations. The growth rates between 0-5 and 5-10 weeks were calculated and graphed as a linear plot in Excel. Student’s t-tests were run to compare the 0-5 and 5-10 week growth rates for all yeast/whole-wheat combinations. Mathematical Transformation: Intensity of yellow color was determined as A420nm in a Hitachi spectrophotometer and converted to beta-galactosidase activity units using the following formula.
with time (10) in minutes, volume(0.75) in mL of reacted lysates, 1.75 is the Molar extinction co-efficient of ONP, A600 represents the absorbance reading at 600nm of 1:10 diluted unreacted, whole cells (to account for differences in cell density) and A550 is measured in the halted reactions at the same time as A420nm measurements were taken( to account for turbidity from cell debris).
