It is a lab experiment ( I WANT YOU TO DO THE LAB REPORT)

timer Asked: Nov 26th, 2018
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Question description

Hey I will upload three files the first one is lab manual and the second one is powerpoint slide to help you in doing the report and answer the question. Then, I will also upload the 1 Worksheet for the lab, which you will use to write the report on. also the last two are the rustle my group number is 8

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

Tutor Answer

School: UIUC

fine.. Expect a good job as always..

Biology 115 Lab Fall 2018

Lab 8 Worksheet


I. Student Name:
Group Members:

[0.5 pts]
DNA Analysis From DNA To Protein – Transcription And Translation
[0.5 pts]
1. The bacterial cells with the plasmid and the ampicillin resistance gene will grow and will fluoresce in
the presence of ultraviolet light


IV. Introduction
IVA. General Theory/Background

This lab included the use of the important techniques which allow manipulation of DNA in order to alter
genes of specific cells. Escherichia coli is an organism which is used for genetic transformation due to its
features that allow it to be easy to work with in the lab. Therefore the aim was to transform bacterial
cells with plasmid DNA and then observe the new phenotypic traits that were attained from the genes
that were conveyed from the plasmid.

Page 1 of 9

Biology 115 Lab Fall 2018

Lab 8 Worksheet

IVB. Summary of Overall Approach



Once the cells are transformed, the presence of the introduced gene will be confirmed through the use
of polymerase chain reaction (PCR) and agarose gel electrophoresis.
PCR is used to make multiple copies of a portion of DNA through the process of DNA replication. The
components of a PCR reaction mix include a template, a primer to begin a new DNA strand,
deoxyribonucelotides, and a DNA polymerase. This process can be carried out in a test tube. The three
steps in PCR are denaturation (separating DNA), annealing primers, and extension. In the first step,
double-stranded DNA is heated to 94ºC, so that the hydrogen bonds can be broken, which causes the
two strands to separate and become templates for the creation of new DNA strands. During the second
step, the temperature is lowered to 55ºC and the primers anneal to DNA complementary sequences on
the template strands. In the last step, an enzyme called Taq polymerase is used to extend the primer by
bringing complementary nucleotides which make phosphodiester linkages. This builds a complementary
strand to the template strand. The reason why this specific enzyme is used is because heat is added in
this step, and heat causes many enzymes to denature. However, Taq polymerase is found in bacteria
that live in hot springs and therefore functions at high temperatures
Procedure_ part 1: PCR Reaction

We put on glove to avoid contaminating the PCR mixtures
Tubes were kept on ice
Used new tip for each addition
Added reagents to the tubes as shown below
Tube 1 (ul)
Tube 2 (ul)
Plasmid DNA
Forward primer
Reverse primer
Master mix
Total volume

5. The tubes were then span for 15 sec
6. The tubes were then placed in the slots of the PCR machine and labeled.
7. The tubes were then retrieved from the machine and placed back in their rack in ice bucket
Page 2 of 9

Biology 115 Lab Fall 2018

Lab 8 Worksheet


Procedure – Part 2: Agarose Gel Electrophoresis
The PCR products were run on 0.8 agarose gel in order to visualize DNA fragments on a UV
1. Obtained 2 sterile microfuge tubes
2. Added 10 ul of the PCR reaction to labeled tube
3. Added 2 ul of 10X agarose gel loading dye and glycerol
4. Loaded the gel in the order of: lane 1; 10 ul sample
Lane 2: 10 ul negative control
Lane 3 : empty
5. Covered the gel and connected the electrodes
6. Gel ran for at least 30 minutes at 125V
7. When electrophoresis complete, gel visualized on trans-illuminator

Procedure :
Bacterial transformation:
Begin by taking two microfuge tubes of “transformat1ion solution,” which contain cold 50mM CaCl2 to
in orde...

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