Biology 115 Lab Fall 2018 Lab 8 Worksheet Instructors: _____________________________________________________________________________________ I. Student Name: Group Members: _____________________________________________________________________________________ II. Personalized Title [0.5 pts] _____________________________________________________________________________________ III. Hypothesis [0.5 pts] 1. _____________________________________________________________________________________ IV. Introduction IVA. General Theory/Background [1.0 pts] IVB. Summary of Overall Approach [1.0 pts] Page 1 of 4 Biology 115 Lab Fall 2018 Lab 8 Worksheet Instructors: _____________________________________________________________________________________ V. Observations & Results VA. Data [0.5 pts] VB. Calculations [0.5 pts] Page 2 of 4 Biology 115 Lab Fall 2018 Lab 8 Worksheet Instructors: _____________________________________________________________________________________ VI. Discussion Questions 1. What is meant by bacteria transformation? [0.5 pt] 2. Bacteria transformation occurred in which agar plate (s)? Explain? [0.5 pt] 3. In this experiment, what effect would we see on LB/Amp/IPTG/Xgal plates if the lacZ gene of pUC18 was interrupted? Why? [0.5 pt] 4. What would be the effect on PCR reaction if the Tm is too low? How will this affect the results? [0.5 pt] 5. True or false. Human DNA polymerase can be use in PCR. Explain your answer (0.5 pt) 6. In PCR reaction we included a tube without DNA. Why this is needed? Is not this waste of material? (0.5 pt) Page 3 of 4 Biology 115 Lab Fall 2018 Lab 8 Worksheet Instructors: _____________________________________________________________________________________ VII. Conclusion(s)/Inference(s): [1.0 pt] _____________________________________________________________________________________ Total Score: _____ / 8.00 (Includes _____ Bonus Points for ________________________________________________) Comments: ___________________________________________________________________________ _____________________________________________________________________________________ Page 4 of 4 Quiz Time! Please hand in your Lab 7 Report You have 5 minutes (from 2:10 to 2:15 pm) to complete the quiz 1. Silence your cell phone and put it away 2. Have your calculator available in case you may need it for the quiz 3. Work on your own; do not cheat 4. When done, turn your quiz upside down and raise your hand for a TA to come and collect your quiz 5. Keep quiet until all quizzes have been collected and counted Schedule November 12, 13, 14 & 15 Exercise 8: Nucleic acids (Lab Report 7 Worksheet Due) November 19-25 No Lab, Thanksgiving (Lab Report 8 Worksheet Due) November 26, 27, 28 & 29 Exercise 9: Control of cellular function (Osmosis diffusion) (Lab Report 8 Worksheet Due) December 5 (Wednesday) Final Exam (Exam 2) Final Lab exam Wednesday December 5th Gowan Hall Rm. 126 12:40 pm to 1:30 pm (during your regular lecture time) Exam will cover the material discussed in the lab, lab manual and PowerPoint slides Cumulative PCR (Polymerase Chain Reaction) • A very quick, easy, automated method used to make copies of a specific segment of DNA • What’s needed…. 1. DNA template 2. DNA primers that identify the desired sequence to be cloned (PCR amplify area between primers) 3. Heat-resistant DNA polymerase Taq DNA polymerase (does not denature) 4. DNA nucleotides (DNTP) 5. Thermocycler 6. Buffer • Amplifying DNA :in vitro Polymerase chain reaction (PCR) 3 steps/cycle 1- Denature (94 C) DNA strands separation 2- Annealing (50-58 C) Primers bind to DNA 3- Synthesis (74C) DNA polymerase Primer: DNA polymerase amplify area between the primers 2 20 6 10 PCR Applications • Genetic testing - Diagnose disease, identify mutations/genetic carriers - Tissue typing for organ transplant - Identify cancer types & customize therapies • Identify new virus or bacterial strains • Forensic DNA testing – genetic fingerprinting • Research applications: - Gene expression levels - DNA cloning - Mutagenesis (random and site-directed) Today’s PCR Experiment 1. Be sure to wear gloves to avoid contaminating your PCR mixtures 2. Work on ice to prevent nonspecific priming (multiple nonspecific products) 3. Remember to use a new tip for each addition, even if it is the same reagent. 4. Add the reagents to the tubes as indicated: Materials Tube 1 Plasmid DNA 2 µl H2O Tube 2 Tube 3 2 µl 2 µl Forward primer Reverse primer H2O 2 µl 2 µl 2 µl 2 µl 2 µl 2 µl 6 µl 6 µl 6 µl Master mix 10 µl 10 µl 10 µl Total volume 22 µl 22 µl 22 µl (one tube/student) 6. Spin tubes for 15 sec. 7. Bring your tubes (still in the ice bucket, WHY?) to the PCR machine. • Tube 2 and 3 are negative control. Very important in PCR (WHY?) • Master mix contains, DNTPS, buffer and enzyme Gel Electrophoresis 1. A method of separating mixtures of large molecules (such as DNA fragments or proteins) on the basis of molecular size and charge. 1. How it’s done • An electric current is passed through a gel containing the DNA • Molecules travel through the gel at a different rates according to: 1- Voltage 2- Size Larger slower 3- Gel concentration high concentration for small DNA fragments & low concentration for large DNA fragments 4- Shape (plasmid), Supercoiled, nicked, linear • Restriction fragments analysis Gel electrophoresis Ethidium bromide DNA has negative charge DNA Loading Dye • DNA samples are combined with 6x loading dye • contains xylene cynanol FF (XCFF) and bromophenol blue (BPB) for tracking sample during gel loading and electrophoresis • contains glycerol to make the sample sink to the bottom of the well • EDTA to inhibit metal-dependent nucleases • DNA Loading Dye: In 1% agarose with TAE buffer, XCFF migrates similar to ~4kbp DNA and BPB migrates similar to 500bp DNA Visualizing DNA • Ethidium bromide (EtBr) fluoresces under UV light when bound to DNA • Allows DNA bands to be visualized Beware! Because of it’s interaction with DNA, EtBr is mutagenic UV Transillumination • You should wear gloves throughout the electrophoresis procedure • Prepare PCR samples for electrophoresis - Spin PCR tubes, 10 seconds - Combine 10µl of each reaction with 3 µl 6x loading dye - mix, spin 10 seconds - Load samples into gel Tube 1 Tube 2 Tube 3 3µl 3µl Label Add loading dye (blue & dense) 3µl DNA Ladder/marker: Is a set of standards that are used to identify the approximate size of a molecule run on a gel during Electrophoresis. Central Dogma of Molecular Biology Transcription DNA Translation RNA • Genotype • Specific DNA sequence • Phenotype • Observed trait Protein Vector: DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA Ampr Ori pBR322 4361bp Plasmid (vector) Tetr • Plasmids are double-stranded circular DNA, in the cytoplasm of a bacterium or protozoan, has the ability to replicate even if the host cell is not replicating. LacZ MCS • Plasmids are the means by which cloned DNA is often transferred into the bacteria. Vector/Insert size pUC18 Ori Ampr for cloning Plasmid characteristics A- Origin of replication Ampr Ori pBR322 Determine plasmid number/bacteria. Low copy number (~20), high copy number (~200) 4361bp Tetr B- AB resistance site Is the means by which antibiotic resistance is often transferred to bacteria to another. LacZ MCS C- MCS Used to cleave plasmid by restriction enzymes, leaving sticky or blunt ends. To insert gene of interest. Circular 3 shapes pUC18 Ori Ampr Transformation: Insertion of Recombinant Plasmids into bacteria (competent cells) Competent cells CaCl2 Glycerol Only some of the bacteria take up a plasmid—How do you know which ones did? * SELECTION using antibiotic resistance Will not Grow in presence of AB Grow in presence of AB Selection (Elimination of non-transformed bacteria) Identification of cells containing plasmids • Selection of recombinant plasmid • Bacteria cells containing plasmids (recombinant or wild type) become ampicillin (AB) resistant. • Grow cells on medium containing (AB) ampicillin Detection of recombinant bacteria vs, wild type vector α-complementation (the blue white screening) • Detection of recombinant bacteria (white colony) vs. only vector/WT (blue colony) • Competent bacteria carries mutant lacZ–ω Plasmid carry lacZ-α • Both form B-galactosidase enzyme, produce blue color in presence of X-gal • MCS within plasmid lacZ-α p 5’ 3’ Add IPTG to enhance the lac operon 5’p 3’ Phosphatase Today’s Experiment 120 l competent bacteria A Add 5 l H2O B 5 l plasmid Incubate ALL tubes on Ice for 30 minutes Heat shock ALL tubes at 42°C for 1 min Incubate ALL tubes on ice for 2 min Add 955 l warm LB to both tubes 990 l LB Repeat 10 l A A A B A B C D LB-agar + Xgal + IPTG LB-agar + AMP+ Xgal + IPTG LB-agar LB-agar + AMP Spread 10 l /plate Incubate overnight at 37°C (Next Day) Count colonies and observe colony color Remember you plated only 10 l of the 1000 l (dilution factor) Plating Technique by Spreading Monday Section Group 1 Tube # 1 2 Group 2 3 1 2 3 Group 3 1 2 3 Group 4 1 2 3 Monday Section Group 5 Tube # 1 2 Group 6 3 1 2 3 Group 7 1 2 Group 8 3 1 2 3 Lab 8-Transformation Result Monday Section Monday- Group 1 Monday-Group 2 Monday-Group 3 Monday-Group 4 Monday-Group 6 Note: there are two group 6 in Monday section Monday-Group 6Ben….mentioned on tape Note: there are two group 6 in Monday section Monday-Group 7 Monday-Group 8 
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