Lab Report 1 Guide Abstract (10%) -objective of experiment (one sentence) -brief summary of experiment (digestion, agarose electrophoresis, and restriction map construction.) -state results of experiment (which plasmid?) Introduction (25%) -discuss all concepts for each experiment, technique. (don’t just report what you did in lab, generally describe these concepts) (restriction enzyme, agarose gel electropherasis) -must have references -chemical reactions and mathematical equations (ethidium bromide, log plot of DNA ladder) Methods (15%) I highly recommend you read a scientific journal article. -Write out procedure but make it as short and concise as possible -Write it in the passive voice (use the zombie test if unsure—see the Lab report information sheet in the Course information section, general guidelines #6). -Make sure to include the concentration of all buffers and reagents -Do not list the reagents you used before the procedure. -Do not copy and paste it from your protocol. -Do not include information that a scientist would already know how to do. -how to make dilutions, set up a reaction, or make buffers. -how to make a gel or set up a column. -Do not put data tables in your methods section. Example 1 This shows the volume and concentration of each reagent Digestion A single digest was done by adding 5µl of pBluescript plasmid (200ng/µL) to 2µL of Xba I (1U/µL) and 2µL of 10X digestion buffer (1.5 M NaCl, 200mM Tris-HCl pH 7.0) in a total volume of 20uL. A double digestion was done as above with the addition of 1.5µL of Hind III (3U/µL). All reactions were incubated at 37°C for 1hr and run on a 1% agarose gel at 80V. Or, you can be even more concise Example 2 This shows the total amount of each reagent by multiplying the volume and the concentration ie. 200ng/uL x 5uL = 1000ng of total DNA in the reaction Digestion The pBluescript plasmid (1000ng) was digested for 1hr at 37°C with Xba I (2U) alone or with Xba I (2U) and Hind III (4.5U) in 1X digestion buffer (150mM NaCl, 20mM Tris-HCl pH 7.0). Samples were analyzed via gel electrophoresis (1% agarose at 80V). Results (20%) -Include all graphs, gels, calculations, one sample calculation is fine. (gel picture, table to summarize all bands in the gel and distance measured, log plot, reconstructed plasmid map) -Also need to briefly describe results in words. (What does the gel show?). -Make sure all figures are labeled properly and have titles -While there are several programs/websites available that will draw a plasmid for you based upon the information you give it, you MUST DRAW YOUR OWN PLASMID MAP! This is a learning exercise for you. I am perfectly confident that programs such as SnapGene can draw a map for you but you will learn very little or nothing by using such a program. Please use a simple drawing program, make a circle and add hash marks for your restriction sites. Example Fig 1. PCR of Gemin2 Gene 1 2 Cntl 872bp 603bp 03bp a) b) Fig 1a. 1 % Agarose gel of PCR Reactions. The gemin2 gene was amplified from Hela S3 cDNA with two different sets of primers (one with and with out a stop codon). PCR reactions were run on gel to determine if the correct products were present. Lane 1 shows the amplification of the Gemin2 gene with a stop codon, Lane2 with out a stop codon, and Lane 3 is a control amplification of the B actin gene. Fig 1b. shows the DNA mw standard fragments that were run on the gel in a) and the corresponding base pairs for each fragment. All three reactions show amplified bands of the correct size, 850bp for the gemin2 gene and 660bp for the B actin gene. Also in lanes 1 and 2 there was a band that was smaller then 72bp which was due to the unused primers in the PCR reaction. Other then the unused primers in the reaction there were no other DNA bands from non-specific PCR amplification in these reactions. Discussion (30%) -Interpret your data. (how did you deduce the restriction map from the gel?) -Were your results as expected and if not, why? (be reasonable) -Refer back to the specific figures in the results section Example Discussion The results of the digestion and agarose gel clearly show that there is only one XbaI cut site on the plasmid. First of all there is only one fragment in the XbaI single digest lane, which is indicative of a linearized plasmid (Fig 1 lane 3). Furthermore, because the XbaI digested fragment is 4.5kb compared to the 3.5kb fragment of the undigested super coiled plasmid (Fig1. Lane 1), it shows that the XbaI enzyme did actually cut the plasmid. The XbaI enzyme cut the super coiled plasmid, which allowed the DNA to unwind, linearize and consequently the 4.5kb traveled more slowly through gel. -Limitations of experiments (Please do not include human error). -Questions from protocol Plasmids • Circular, extrachromosomal DNA  Restriction mapping of plasmids Bacterial “accessory” chromosomes • Can be passed from one bacteria to another Ability to transfer genes  Importance: antibiotic resistance  • Generally small: ~5-10 kBP Plasmid features • Origin of replication  Determines host • E. coli plasmid must contain an E. coli origin  Determines low-copy or high-copy • Low = 1-2 copies/cell • High = 100’s • Genetic marker  Antibiotic resistance gene • Restriction sites Restriction endonucleases • Cleavage of dsDNA at specific base sequence   Degradation of foreign DNA Host DNA protected by specific methylation • Restriction sites   Palindromic, 4-8 bp Sticky ends or blunt ends • EcoRI: 5'-G-A-T-C-C...3' 5'...G-G-A-T-C-C...3' 3'-G...5' EcoRI 3'...C-C-T-A-G-G...5' 5'...G-3' 3'...C-C-T-A-G-5' 1 Restriction sites • Uses of restriction enzymes:  Cleavage of large DNA molecules pBR322 • Two antibiotic resistance genes  • DNA fingerprinting  Transfer of genetic material • Hybrid DNA molecules  Restriction maps pUC18  Different restriction sites in each gene Addition of foreign DNA (cloning) into ampr gene: tetracycline resistance only pUC18 • β-galactosidase gene (lacZ) and ampr   Cloning into lacZ gene: no β-galactosidase production Use of X-gal in media • X-gal cleaved by βgalactosidase, then oxidized: blue 2 Plasmid restriction mapping • Displays restriction sites on a plasmid Agarose gel electrophoresis • Separation of nucleic acids   • Anode is + so DNA migrates to it! • 1. Digest DNA w/ RE’s 2. Separate fragments on gel 200-50,000 base pairs Electric field D-galactose β(1,4) 3,6-anhydro-Lgalactose α(1,3) 100 monomers, ~10kDa Agarose gels • H-bonding within polygalactose units initiates gel formation • Uniform pore size, controlled by agarose concentration % w/v Range agarose (kb) 0.3 5-50 0.5 2-25 0.7 0.8-10 1.2 0.4-5 1.5 0.2-3 2.0 0.1-2 Gel running • Dyes:  Bromophenol blue: migrates ~500 bp • MW standards 3 Gel visualization and measurement SYBR Safe • Ethidium bromide    • Better staining of DNA • Cost comparison: Intercalates between stacked bases of dsDNA Fluoresces under UV Powerful mutagen!   ~$0.40/gel ($0.006/gel for EB) Disposal: • SYBR Safe gels can go in trash (so the manufacturer claims) • EB treated gels must go to hazardous waste at a cost of about $150/year Building a plasmid map • Restriction enzymes:    E H S E+H Building a plasmid map E+S H+S EcoRI (E) HindIII (H) SalI (S) E 5 4 3 2 H Next, use E+H The 3 fragments are 3 kBP, 1.2 kBP and 0.8 kBP S E+H E+S H+S 5 4 3 This means the 3.8 kBP fragment from H has been cut 2 H 1 1 0.8 Start with the single enzyme that cuts twice (H) The 2 fragments are 3.8 kBP and 1.2 kBP 1.2 H E 0.5 0.5 5 kBP 5 kBP H H 3.0 4 Building a plasmid map E H S E+H Next, use H+S The 3 fragments are 3.8 kBP, 0.8 kBP and 0.4 kBP Building a plasmid map E+S H+S E 5 4 This means the 1.2 kBP fragment from H has been cut We still don’t know the relationship between E & S S 2 E 1 0.8 0.5 E S S? 5 kBP 2 H 0.8 0.5 H+S 3 1.2 S? E+S 4 This gives us the location of S and our finished map: 1 H E+H 5 3 0.8 3.0 H Finally, use E+S The 2 fragments are 3.4 kBP and 1.6 kBP 0.4 H 5 kBP H 3.0 5 BCH 467: Lab #1 Restriction Map of DNA Introduction: What color is your DNA? You will be assigned to investigate an unknown DNA sample: A or B. The DNA contains a gene encoding either a red fluorescent protein (RFP) or a green fluorescent protein (GFP). In the first experiment, the DNA will be cut and the resulting fragments separated by agarose gel electrophoresis. From the sizes of the fragments, you should be able to piece together a “map” of the DNA. Next week, you will determine the sequence of a portion of the DNA. By comparing the DNA sequence to the amino acid sequences of the red and green fluorescent proteins, you should be able to identify the gene. In the third experiment, you will use a diagnostic PCR test to identify your gene. Combining all of these results should allow you to give a detailed description of the DNA. Background information on these types of procedures is covered in Chapters 11 and 12 of the textbook (Ninfa and Ballou, 1998, Fundamental Laboratory Approaches for Biochemistry and Biotechnology, pp. 277-299 and pp. 313-323). A general introduction to fluorescent proteins is given in the article: Matz, M. V., Lukyanov, K. A., and Lukyanov S. A., 2002, “Family of the green fluorescent protein: journey to the end of the rainbow”, BioEssays 24, 953-959. More details on the biochemistry of fluorescent proteins can be found in the review: Zimmer, M., 2002, “Green fluorescent protein (GFP): applications, structure, and related photophysical behavior” Chem. Rev. 102, 759-781. Restriction Map of DNA In the laboratory, DNA is commonly contained in plasmids, which are small, circular pieces of DNA that can replicate independently of the chromosome. DNA is often manipulated using restriction enzymes, which cut the DNA at specific recognition sites. The sizes of DNA fragments can be determined using agarose gel electrophoresis with visualization of the DNA by SYBRsafe staining. In this experiment, you will use these tools to determine how many sites your plasmid contains for three different restriction enzymes, and how far apart the sites are on the DNA. MATERIALS 1. L20 pipettor, sterile tips, sterile 1.5 ml eppendorf tubes eppendorf tube rack, float 2. Plasmid A or B (50 nanograms/microliter in 1X digestion buffer) You will investigate either plasmid A or plasmid B. Use the same type of plasmid unknown for all of the experiments in the DNA section. 3. 1X digestion buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 100 mM NaCl, 1 mM 2-mercaptoethanol) 4. Restriction endonucleases BamHI, PstI, and ScaI (2 units/microliter in 1X digestion buffer, on ice) (one unit is the enzyme activity that completely cleaves 1 microgram  DNA in 1 hour at 37 °C) Sequences of restriction sites: BamHI: 5’-GGATCC PstI: 5’-CTGCAG ScaI: 5’-AGTACT 5. gel loading solution (30% glycerol, 10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.025% bromophenol blue) 6. DNA size markers (1 kb ladder, New England Biolabs) (50 nanograms/microliter in 0.006% xylene cyanol FF, 0.006% bromophenol blue, 0.06% orange G, 2.5% Ficoll 400, 10 mM Tris-HCl, pH 7.9, 10 mM EDTA) 7. 37 °C incubator 8. 0.5X TBE (44.5 mM Tris base, 44.5 mM boric acid, 1.0 mM EDTA) 9. molten agarose (1% agarose in 0.5X TBE, boiled and then cooled to ~55 °C) 10. horizontal gel electrophoresis apparatus and power supply 11. staining solution Syber SAFE 12. UV imager 2 PROCEDURE 1. Set up restriction digests of the plasmid DNA: Prepare three single digestions and three double digestions with the restriction enzymes BamHI, PstI, and ScaI, and one sample with no enzyme added. Each digestion should contain 250 ng of plasmid and 10 units of each enzyme in the digestion in 1X digestion buffer with a total volume of 20 µl. Use sterile eppendorf tubes and sterile pipet tips to avoid contaminating the solutions with nucleases, and use a fresh tip each time an aliquot is taken from the stocks. Example of digestion setup: 1 BamHI Digestion buffer Plasmid (50 ng/µl) BamHI (2 U/µl) PstI (2 U/µl) ScaI (2 U/µl) 10 µl 5 µl 5 µl 2 PstI 3 ScaI 10 µl 5 µl 10 µl 5 µl 5 µl 5 µl 4 BamHI & PstI 5 µl 5 µl 5 µl 5 µl 5 BamHI & ScaI 5 µl 5 µl 5 µl 5 µl 6 PstI & ScaI 5 µl 5 µl 7 uncut 15 µl 5 µl 5 µl 5 µl 2. Incubate digestion tubes in the 37 °C incubator or heat block for at least 30 minutes. 3. Pour an agarose gel: Insert the gel casting tray into the casting fixture. Slowly pour 50 ml of molten agarose into the gel tray (make sure the SYBRsafe has been added by the TA and WEAR GLOVES). Insert the comb into the slots in the casting tray. Allow the gel to harden undisturbed at room temperature. 4. Set up the gel electrophoresis apparatus: Add 300 ml 0.5X TBE to the main gel box. When the gel is solidified (fully opaque), transfer the casting tray to the main gel box. Remove the comb. The running buffer should just cover the gel. 5. Load and run the samples: When the incubation is done add 5 µl of loading buffer to each sample. Load 20 µl of each sample in a separate lane on the gel. Load 10 µl of the size markers in the remaining lane on the gel. Record the order of the samples in the lanes. Place the cover on the gel box, connect the electrodes, and set the power supply to 100 volts. Electrophorese at 100 volts for 1 hour or until the blue dye is approximately 2/3 of the way down the gel. The size marker contains three dyes: xylene cyanol FF migrates at approximately 4 kb, bromophenol blue at approximately 300 bp and orange G at approximately 50 bp. 6. Stain and photograph the gel: When the electrophoresis is finished, bring the gel to the TA, who will photograph the gel using the Kodak digital gel imaging system. WEAR GLOVES DURING THIS STEP. 3 ANALYSIS: On the photograph of the gel, measure the migration distance from the well to the band for the size markers and plasmid fragments. For the size markers, plot log(size in kilobase pairs) versus migration distance or use a semilog plot. Fit a straight line in the linear region of the size markers, and use this to calculate the sizes of the plasmid fragments. DNA size markers (1 kb ladder, New England Biolabs) This image is for your reference only so you know the sizes of the bands in the ladder. DO NOT MEASURE THE DISTANCES ON THIS IMAGE. Using the fragment sizes determined from the analysis of the gel photograph, determine the restriction map of your plasmid. Show the distances between the restriction sites of the three enzymes. The plasmid can be thought of as having two parts: the vector and the insert. A vector is a plasmid that has been engineered to be able to “carry” pieces of DNA. Different genes can be inserted into restriction sites on the vector. The vectors used for the fluorescent protein genes are called pRSETB and pQE30. The pRSETB vector contains 1 ScaI site, and the pQE30 vector contains 2 ScaI sites. The fluorescent protein genes are in the inserts, which do not contain ScaI sites. Before the next lab period, you should determine whether your vector is pQE30 or pRSETB. 4 Lab reports must be submitted to SafeAssign through Blackboard. Please include your name and email address at the top of the first page of your report. Do not include your student ID number. Any equations and math should be typed using a program such as Mathtype or Microsoft Equation Editor. All figures should be numbered, clearly labeled and must be referenced in the text. The file you submit must be a PDF file with a size limit of 2 MB. You must adhere to the page limits any text beyond the page limit will not be read. LAB REPORTS MUST BE TYPED IN 12 POINT FONT AND DOUBLE SPACED! Sections of the Lab Report: Title (you do not need a title page) Abstract (10%) 1. Brief project description 2. Most important results and conclusions Introduction (25%) 1. Objective or purpose of the experiment (what are the goals?) 2. Brief discussion of theory behind the experiments and techniques 3. Any chemical or biochemical reactions 4. Any equations used for analysis of data Materials and Methods (15%) 1. A narrative of what you did in the lab. In order to write this section, you need to read a journal article (Journal of Biological Chemistry is a great example). 2. Should include all reagents and instruments used in the experiment. 3. Should enable the reader to repeat your experiment and arrive at similar results 4. Should be clear and concise 5. DO NOT INCLUDE A LIST OR TABLE OF MATERIALS Results (20%) 1. An opening statement to describe the general results obtained 2. Raw data (tables, graphs, photos of gels, etc.) in an organized manner and clearly labeled figures must be scanned and pasted directly into the text 3. Graphs must be generated with a program such as Excel or Sigma Plot 4. Calculations (refer back to equations mentioned in introduction) 5. Factual description of results 6. Figures and tables must have a title and description Discussion (30%) 1. Significance of results 2. Discussion of unexpected results, problems encountered, failure of experiment 3. Interpretations supported by data 4. Limitations of data 5. Suggestions for improvement 6. Questions from text References (may be single spaced) 1. Citations from text 2. Other literature While there is no grade for this section, lack of references will impact the introduction section Some general guidelines for lab reports: 1. Italics, symbols (w) and sub/superscripts need to be used when appropriate. 2. Don't refer to each week's experiment as a separate week. Think of the lab as one big study and present it in a logical manner (not necessarily based upon the order in which you performed the experiments). 3. When using an abbreviation, write out completely one time, then include the abbreviation in parentheses immediately after. After that, you may use the abbreviation 4. Avoid excessive use of capitalization. Most science related words (such as polymerase chain reaction, affinity chromatography, imidazole, etc...) are not capitalized. 5. When reporting standard deviation, it should be done in a manner similar to the following: 0.567 0.023 mg/ml (note that the units come after the error) 6. The pronouns "T" or "We are not used! The verb tense is always the same. In science, the passive voice is used, especially in the materials and methods section. If you are not sure whether or not you are writing in the passive voice, use the zombie test. You should be able to add the phrase ...by zombies." to the end of any sentence. As an example of something that passes the zombie test: 10 ul of buffer was added to each of the reaction tubes (by zombies). This would not pass the zombie test: I added 10ul of buffer to each reaction tube (by zombies). Use the zombie test and you should never lose points for failing to use the passive voice. 7. Each sentence should express a single thought and be complete without being a run-on sentence or having other ghastly grammatical errors. 8. Each paragraph should cover one topic. If you are putting multiple topics into the same paragraph and that paragraph becomes rather lengthy, you should start a new one. 9. All figures are to be included in the appropriate section. Appendices are not allowed and will be disregarded by your TA. 10. A good way to write your results and discussion sections is to start by preparing all of your figures. Then, write these two sections around your figures.
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