Materials and Methods: Plasmid Isolation and Transformation
Please write up the plasmid isolation and transformation procedures done in lab in the format of a research report's Materials and Methods section. This assignment is worth 15 points and is due by 5 pm on Wednesday Oct. 14th for all students.
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. Where possible, use references to previous published descriptions of common protocols rather than describing the methods yourself (such as the Inoue method of making cells competent). To see how this is done properly, look at Materials and Methods sections from published papers. Do not clutter your paper with trivial or non-essential 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 the tool, Gel Electrophoresis, or a vague future goal, 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 precipitated 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. Your 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. Do NOT use ambiguous or meaningless terms such as Solution I,II, or III. The reader needs to know how to make those stock solutions by being told the ingredients and concentration. However, in most of a protocol description, rather than giving recipes (e.g. mixed this vol. of this with this vol. of that), wherever possible, 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 0.1M Na2CO3 was added to each tube in 30 sec. intervals.
Sample partial Methods section:
Restriction Enzyme Digestion and Separation of Plasmid Fragments by Electrophoresis
Isolated plasmid DNA (from Plasmid DNA Isolation described in Materials and Methods previously) was digested with a restriction enzyme by combining 20% plasmid DNA with 0.4% HindIII (stock diluted according to supplier, SigmalAdrich, at www.sigmaaldrich.com) in High Salt Buffer (0.1M NaCl, 50 mM Tris pH 7.5, 10mM MgCl2, and 1mM DTT) for 20 min at 37C. The DNA fragments from the digest 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.
Note, that it is not assumed that your reader knows from where the starting materials come. You must explain that the plasmid DNA that you are digesting was prepared from a protocol previously described and where you purchased anything else that you don't describe how to make.
Note, also, that in the example above it was not possible to give the preferred effective concentrations of either the plasmid DNA or the restriction enzyme because we don't know the DNA concentration in our plasmid isolate and the supplier of the commercially purchased restriction enzyme, HindIII, did not give the concentration, but did supply working dilution recommendations. Therefore, volume ratios is about the best we can do. Note that it would be possible to figure out the effective concentration of the ingredients in the buffer but that is necessary since the buffer is the diluent. The reader only needs to know how to make the stock.
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.
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.
Grading Rubric for Materials & Methods – 15 points
|At or Above Standard
|2-3 pts. Protocols divided appropriately and titled by experimental goal rather than tool or method
|0-2 pts. Protocols not divided into appropriate sections and/or not titled by goal
|5-6 pts. Explained and identified clearly, completely and succinctly how a new investigator could obtain or make the starting materials and progress from those materials to the end goal. Did not include trivial detail; did not use “tube talk” or “protocol speak”. Did not use informal language (such as “miniprep”)
|0-4 pts. Did not correctly identify or explain how all materials were made or obtained. Did not explain clearly or succinctly how the starting materials were converted (in a stepwise, defined progression) to intermediate stages and eventually to the final product. Used “tube talk” or “protocol speak” rather than explaining the experiment progression. Used colloquial language. Focused on lab tools or chronology of lab experience (“did this; then did that” approach).
|Accuracy of information/
Appropriateness of information
|5-6 pts. Accurately described the protocols, giving effective concentration where indicated. Included conc. and ingredient information for all stock reagents or enough information to locate source of proprietary reagents. Started and ended at appropriate point for each protocol.
|0-4 pts. Did not accurately describe one or both protocols. Did not calculate effective concentration, didn’t calculate it correctly, didn’t use it where appropriate, or calculated it correctly but didn’t write about conc. in language that made it clear the reference was to effective rather than stock concentration. Did not include conc. and ingredient information to make stock reagents or give the source and enough information to buy proprietary reagents. Did not start or end protocol at appropriate step.