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Keywords:

  • Plasmid preparation;
  • problem solving;
  • professional skills

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES

This laboratory exercise encourages upper level biochemistry students to build and expand upon previously developed laboratory skills and knowledge as they conduct a comparison of two methods of plasmid preparation based upon cost, quality of product, production time, and environmental impact. Besides creating an environment that mimics a more realistic practice of science, there are several key learning objectives. First, students will learn to effectively plan and manage time so that they can meet a scientific goal. A second objective is for them to learn to use theory as a basis for understanding new and different technology since classroom exposure is necessarily limited. Third, they will learn to think critically and logically to solve problems, and they will communicate this in written format. All of these skills are particularly important in preparation for a scientist's professional life.

The ability to isolate and characterize biomolecules involves several basic skills in which all biochemistry and molecular biology students should be proficient. Among some of these skills are plasmid isolation and mapping and the use of spectroscopy and electrophoretic techniques [1]. Although this laboratory exercise seeks to help students develop technical skills, it also has the additional goal of helping them with professional development and the acquisition of skills and competencies needed beyond the classroom. Those skills developed in this laboratory exercise include the ability to develop and implement plans to achieve a goal, to work cooperatively, to think critically and logically to solve problems, and to communicate persuasively and effectively in writing.

Development of proficiency in technical skills often requires repetition. In most classroom laboratory settings, students usually only have a single opportunity to carry out a procedure, and quite often, it is a first attempt at performing such a procedure. As is customary, initial outcomes are often not the best, and it can be frustrating for both the students and the instructor. Nor does such a situation reflect the actual practice of science where a researcher often has to repeat and refine a procedure several times to get satisfactory results. Although there is certainly not the time to repeat every procedure as often as necessary, there is great value in allowing students to repeat procedures. Most importantly, repetition allows them to critically evaluate their methods and then make appropriate adjustments to improve their initial results. In an effort to more closely simulate an actual laboratory setting, this laboratory exercise is designed to allow students to repeat a plasmid preparation procedure while also learning how to isolate plasmid DNA using a commercial kit. The chemistry of this kit is based upon that of alkaline lysis; students are expected to be able to see the parallels so that they can understand how the kit works. Besides enabling students to apply theory to new and different situations, the introduction of commercial kits is another way to mimic a real-world situation in which a person must be able to perform a task simply by following a written procedure that s/he may or may not be familiar with.

After isolation of plasmid by each method, the students perform spectroscopic and restriction enzyme analyses to determine the purity and identity of the plasmid. Finally, they are asked to compare the procedures based upon the following parameters: yield of product, cost, production time, and environmental impact. Practicing scientists in any setting are continually concerned with reaching a scientific goal within a set time frame and budget. Students do not often get exposure to this aspect of science during laboratory classes, but given the importance of being cost-effective, they need to develop such skills.

Concern for the environment has become a major issue for the current generation of scientists and will undoubtedly be an issue for future generations as well. The liberal arts curriculum at Goucher College has been redesigned to encourage students to explore the ecological and environmental dimensions of their education. Toward that end, our students are encouraged to make choices, when possible, that minimize harm to the environment and conserve resources.

EXPERIMENTAL DESIGN

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES

Materials/Equipment—

The following major equipment is required for this laboratory exercise:

  • UV-visible spectrophotometer

  • Ultracentrifuge (clinical centrifuge may be substituted)

  • Microcentrifuge

  • Horizontal gel electrophoresis apparatus and power supply

  • UV transilluminator

  • Quartz cuvettes

All reagents required for making buffers for electrophoresis and for the traditional alkaline lysis plasmid prep were purchased from Sigma. A mini-prep kit (Qiagen catalog number 12123) containing all necessary reagents was purchased from Qiagen. Restriction enzymes and buffers, DNA molecular weight standards (1-kb ladder), and plasmids pUC18 and pLitmus 28i were purchased from New England Biolabs. Plasmid pBlueScript® II KS+ was purchased from Stratagene.

Safety—

The most significant safety issues for this laboratory exercise are those associated with ethidium bromide (EtBr) and the phenol/chloroform mixture (1:1 v/v), all of which must be treated as hazardous waste. EtBr is a mutagen, phenol is toxic and caustic, and chloroform is a possible carcinogen. The laboratory requires only about 2 ml of the mixture per pair of students. All of the above chemicals should be handled with gloves and goggles; in addition, any use of the phenol/chloroform mixture should be done in a fume hood. Any item that has come into contact with EtBr, as well as gels containing it, should be disposed of as hazardous waste. Electrophoresis buffers that contain EtBr at a concentration of 0.5 μg/ml or less can be decontaminated by stirring overnight with activated charcoal (0.3 g/100 ml of solution). After filtering, the decontaminated solution should be disposed of in the appropriate drain; the filter and charcoal should be disposed of as hazardous waste. UV blocking goggles should be used when viewing gels under UV illumination. Any unused cell cultures should be treated with bleach for at least 1 h and then disposed of in the drain.

Experiment—

Students worked in pairs and were given ∼25 ml of a saturated culture containing cells that had been transformed with pUC18, pLitmus™ 28i, or pBlueScript® II KS+. (Any high copy number plasmids that are well characterized can be substituted for those listed above.) Students were asked to isolate plasmid DNA using both the traditional alkaline lysis procedure and the Qiagen mini-prep procedure. They used 10 and 5 ml of cell culture, respectively. Detailed procedures for the traditional alkaline lysis have been described previously [2], and detailed instructions for the Qiagen procedure are included with the kit.

Upon completion of the procedure, students were expected to report a yield and to identify their particular plasmid by performing restriction enzyme analysis and agarose gel electrophoresis. They were given a list of the three possible plasmids they could have. Single restriction enzyme digests were typically performed on ∼1 μg of plasmid in a volume of 20 μl as follows:

  • 1–3 μg of plasmid (usually 1–3 μl)

  • 2 μl of appropriate 10 × restriction enzyme buffer

  • 1 μl of restriction enzyme (5–10 units)

  • distilled H2O to a final volume of 20 μl

Restriction enzyme was always the last component to be added. The plasmid was digested at 37 °C for 1 h. When double digests were performed, digest conditions were optimized for the first digest. As long as the second restriction enzyme had at least 50% activity under the initial conditions, the digests worked well since an excess of enzyme was used. Double digests were done in a total volume of 30 μl as described below.

  • After the first digest was complete, the following was added to the 20-μl digest reaction:

  • 1 μl of the appropriate 10 × restriction enzyme buffer

  • 1 μl of restriction enzyme (5–10 units)

  • distilled H2O to a final volume of 30 μl

Analysis of the restriction digests was done by gel electrophoresis. Students ran a 1% mini-gel in Tris borate/EDTA electrophoresis buffer with ethidium bromide (0.5 μg/ml) and the appropriate molecular weight markers. The composition of Tris borate/EDTA electrophoresis buffer can be obtained from the laboratory manual of Sambrook and Russell [3]. The running buffer should also contain ethidium bromide at the same concentration as the gel. Gels were run at a voltage that corresponded to 5–8 V/cm. Students who require extra reading on electrophoresis can use one of the standard biochemistry texts, Fundamentals of Biochemistry: Life at the Molecular Level [4], Biochemistry [5], or for more detail, a monograph in Methods in Enzymology [6].

The students should be able to complete both the isolation and the analyses within two 4-h laboratory periods; isolation and quantitation by UV as well as planning of the restriction enzyme digest are typically done during the first week. Restriction enzyme analysis and gel electrophoresis can be done during the second week. The written report was to be completed the following week; although students collaborated in the laboratory, the report was an individual effort.

DISCUSSION TOPICS

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES

Upon approaching this laboratory exercise, students had already performed a traditional alkaline lysis and had also read the original paper describing the procedure [7]. Previously, the students had turned in a short paper in which, among other things, they had to write an extensive discussion about their previous plasmid isolation. Students commonly encountered the following problems with traditional alkaline lysis methods: high levels of RNA and/or low yields of plasmid DNA. The students were given the opportunity to troubleshoot, and most were able to draw reasonable conclusions about potential sources of error. For example, students who had high levels of RNA were asked to review their procedures; some realized that they had made errors in calculating the amount of RNase to add, and some concluded that perhaps the RNase was not as active as they thought. This led them to either increase incubation times and/or use a new aliquot of enzyme. Those students with low plasmid yields focused on more complete resuspension of the cells, more complete cell lysis, and/or more careful execution of the procedures so that losses could be avoided. They redirected accordingly and then repeated the plasmid preparation with new and improved procedures.

In addition to repeating the plasmid preparation using traditional alkaline lysis, students perform a plasmid preparation using a Qiagen® plasmid kit. First, as commercial kits are commonly used in academic and industrial research settings, it is beneficial for students who move beyond the classroom to have had exposure to it. Second, it is very common for scientists to have to perform a procedure or protocol that is unfamiliar to them; using a kit provides good practice in understanding and following instructions. Before performing the Qiagen prep, students were asked to read the protocol and troubleshooting guide in the manual that accompanied the kit because they would receive very little guidance from the instructor. The goal was to encourage them to try and understand this procedure based upon what they already knew about the theory behind it. For example, they realized that the solutions that were in the Qiagen® plasmid kit were essentially the same as those used for traditional alkaline lysis. As a result, they understood that the two procedures were essentially parallel until they reached the chromatography step of the Qiagen® plasmid kit.

It was made clear that students would only have 2 weeks to complete the entire project; this encouraged them to understand and plan procedures prior to coming to the laboratory and to work cooperatively during the laboratory period. Traditional laboratories are often well planned out for students, and thus, they need not give much thought to the logistics of carrying out procedures. Providing students with a more open-ended approach places the impetus on them to develop plans to achieve a particular goal and then to implement those plans in a timely manner. During the first week of the assignment, they performed both plasmid preparations and planned the restriction enzyme digests; they were given minimal guidance. In terms of planning the restriction digests, students quickly figured out that they needed to consult internet resources, namely the catalogs and websites of the suppliers, to find maps of the plasmids so that they could design the appropriate restriction enzyme analysis and subsequently interpret the data. Students were encouraged to use sequence analysis software such as Vector NTI® or web-based tools such as NEBcutter found at the New England Biolabs website (www.neb.com). The latter program is free and accommodates a variety of sequence formats or allows sequence retrieval if a GenBank™ accession number is available.

Upon completion of the plasmid preparations, quantitation and restriction enzyme analysis were done. Students were not expected to be familiar with setting up restriction enzyme digests, so they were referred to the technical reference section of the New England Biolabs home page (www.neb.com/nebecomm/tech_reference/restriction_enzymes/setting_up_reaction.asp). Further questions about restriction enzyme analysis were answered during a short discussion held while students were waiting for gels to solidify. They were instructed to perform at least two digests with the only limits being the availability of restriction enzymes in the laboratory. The following enzymes were available and were sufficient for students to carry out analysis: HindIII, EcoRI, SacI, AhdI, AflIII, AclI, and XhoI. After some discussion among themselves, students eventually realized that they would need to determine the size of the plasmid; the easiest way to accomplish this is with a single digest with a unique enzyme. They confirmed the plasmid size either by doing a digest with an enzyme that has multiple sites in the plasmid or by doing a double digest with enzymes that have unique sites. They also included a negative control of undigested plasmid. Fig. 1 shows a gel of typical student results with pBluescript® II KS+. The pBluescript® II KS+ has a size of 2961 bp.

Students were to use their results to compare the two procedures based upon yields and quality of product, production time, cost, and environmental impact. It was emphasized that there were no right or wrong answers; the goal was for them to demonstrate and explain the logic behind their particular conclusion using their data for support. The assumptions were that 50 mini-preps would be performed daily and that all basic chemicals would be purchased from Sigma and/or Fisher Scientific. The assignment required them to make their justification in the form of a short report (no more than 3 pages) to a technical audience. After some discussion, students realized that to make meaningful comparisons, they would need to normalize to a standard (such as plasmid obtained from 10 ml of culture, for example) and that they would need to arrive at a cost per unit (such as cost per 100 plasmid preps). For the most part, students obtained high quality DNA with little contamination from both plasmid prep methods; yields varied depending upon the skill and proficiency of the users. Quantitation of plasmid was performed using UV-visible spectroscopy; a spectrum was run from 200 to 300 nm on an appropriate dilution of the plasmid prep to determine the absorbance at 260 and 280 nm. Absorbance at 260 nm was used for quantitation based upon the standard relationship that when A260 = 1, the concentration of double-stranded DNA is ∼50 μg/ml. Typical student yields ranged from 10 to 50 μg of DNA from a 10-ml prep. Yields from traditional alkaline lysis were routinely higher than those from the Qiagen method. The A260:A280 ratios for all preps were usually 1.8 and higher. Although ratios closer to 2 often indicate contamination by RNA, the gel analysis did not corroborate this (Fig. 1). For the majority of students, both methods gave plasmid that was free of RNA contamination. When there was contamination, it most often occurred with the alkaline lysis method.

The other major aspects of the analysis included production time, cost, and environmental impact. The analyses of time investment, beyond those required for making initial solutions for the traditional alkaline lysis method, were quite different. Students found that they could perform two alkaline preps in the same time it took to perform a single Qiagen prep (2.5 h). For their analysis, students assumed that the bulk of the labor costs would be due to wages and computed these costs by using the current living wage of Baltimore ($9.06/h). Based upon this information, it cost twice as much to produce plasmid using the Qiagen method. The labor costs to produce a single Qiagen prep are $22.65 and $11.33 for a single alkaline lysis prep. Although there is a decrease in the time per prep with simultaneous multiple preps (especially for the Qiagen method), production time will still be less for the traditional alkaline lysis method because of the absence of time-consuming chromatographic steps. Discussions about environmental impact were lively and caused students and this instructor to want to learn more about waste disposal. There was an ongoing debate about the relative impact of plastic disposal versus disposal of chlorinated and non-chlorinated organic wastes. It is certainly an idea for an independent study. All of these factors figured into the final analysis. Student reports came in a variety of formats, all of which were equally effective at justifying their conclusions. Table I shows a sample comparative cost analysis from one student's report.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES

Besides gaining additional practical experience with plasmid isolation and associated techniques for DNA analysis, students gain firsthand experience with the iterative nature of science. They realize that mastery of a procedure comes with practice. For example, most students noticed a significantly smaller investment of time for the second repetition of the traditional alkaline lysis procedure. They also gained a sense of satisfaction because they had a better outcome (higher yields, less contamination) the second time around.

Students also gain exposure to the way in which science is actually practiced. Since most scientists work under time and budget constraints, it is important for students to develop a set of skills beyond the technical skills that are the focus of most undergraduate laboratory courses. For example, this exercise helps students to be able to plan and allocate resources and time to be able to meet a deadline. Students who were successful at this were very adept at maximizing teamwork; they planned out procedures ahead of time and assigned tasks based upon the particular skills of the group members. Those who were less successful needed better interpersonal communication skills as well as more attention to following instructions. This exercise also helps students learn to manage financial resources wisely. By preparing a budget for each procedure and comparing the two, students become engaged in another type of critical thinking that is extremely important for the decision-making process. Lastly, they continue learning the importance of being able to effectively and persuasively communicate.

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Figure FIGURE 1.. Restriction enzyme analysis of plasmid preps. Approximately 1–3 μg of plasmid were digested with the appropriate enzyme(s); each digest was analyzed on a 0.8% agarose gel. Lane A, 1-kb ladder; lanes B and E, undigested plasmid; lanes C and F, EcoRI digest; lanes D and G, EcoRI and AflIII double digest. Students analyzed the entire digest.

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Table Table I. Additional costs for Qiagen preps and alkaline lysis preps
ComponentCostAmount/100 prepsCost/100 preps
All prices are from the Sigma 2004–2005 catalog. TE buffer (pH 8.0): 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0).
    10 mM Tris-HCl (TE, pH 8.0)500g/$42.720.12 g$0.01
    1 mM EDTA (TE pH 8.0)1 kg/$117.500.003 g$0.01
    Isopropyl alcohol200L/$1300.0056 ml$0.36
    70% ethanol16L/$454.00 (100% ethanol)100 ml$1.99
    Total cost for 100 Qiagen preps$439.37  
Alkaline lysis   
    50 mM glucose5 kg/$92.701.08 g$0.02
    25 mM Tris-HCl500g/$42.720.36 g$0.03
    10 mM EDTA1 kg/$117.500.35 g$0.04
    12 mg RNAse A100 mg/$34.3012 mg$4.12
    0.2N NaOH12 kg/$152.503.2 g$0.04
    1% w/w SDS100 g/$78.800.4 g$0.32
    5 M potassium acetate1 kg/$117.5014.7 g$0.54
    10 mM Tris-HCl (TE, pH 8.0)500g/$42.720.12 g$0.01
    1 mM EDTA (TE, pH 8.0)1 kg/$117.500.003 g$0.01
    Phenol:CHCl3400 ml/$190.0060 ml$28.50
    70% ethanol16L/$454.00 (100% ethanol)70 ml$1.99
    Isopropyl alcohol200L/$1300.0060 ml$0.39
    Lysozyme (0.5 mg/ml)1g/$24.9050 mg$1.31
    Total cost for 100 alkaline lysis preps$37.32  

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES

I thank the Goucher College Chemistry department for funds to purchase laboratory materials and supplies. I thank the students in CHE346, in particular, E. Para, whose data appear in Table I, and R. Eisert, whose data appear in Fig. 1.

REFERENCES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL DESIGN
  4. DISCUSSION TOPICS
  5. CONCLUSIONS
  6. Acknowledgements
  7. REFERENCES