I need the lab report and a lab report rough draft
the lab calledImpact of BRCA mutations it's on labster
These are some resources for you to use to create both your final lab report and your mini lab reports.
The LAB for the report: your choice of any labster lab.
(the "Copy of Lab Report..) is an example only.
Copy of Lab Report Template (English).docx
Download Copy of Lab Report Template (English).docx
Lab Report Instructions.docx
Download Lab Report Instructions.docx
SBL Synthetic Biology Lab Manual (English).docx
Download SBL Synthetic Biology Lab Manual (English).docx
(Below is a guide that we will go over to help with how to really view a lab)
https://harford.libguides.com/c.php?g=321391&p=2150319Links to an external site.
Laboratory Report Instructions - Online Writing Lab - Reed CollegeLinks to an external site.
(website)
https://www.reed.edu/writing/paper_help/labreport.html#intro
https://harford.libguides.com/c.php?g=321391&p=2150319
Virtual Lab Report Name of the Simulation This lab report is for you to reflect on what you completed and learned in this simulation, and to practice your written scientific communication skills. Sections Describe the overall objective and make a hypothesis Introduce relevant background knowledge on this topic Summarize the steps taken in the simulation Explain any obtained results Discuss the conclusions and implications 1. Describe the overall objective and make a hypothesis. What is the overall purpose of the experiments or activities? Make a hypothesis if applicable. Hint: The purpose is often stated in the welcome message of the simulation. Write your answers here: 2. Introduce relevant background knowledge on this topic. What have you learned in class or researched on your own that would help prepare for this simulation? Hint: You can review the “THEORY” section in the simulation or at https://theory.labster.com/ if you need help. Write your answers here: 3. Summarize the steps taken in the simulation. Explain each step you completed including the equipment and techniques you used. Hint: You can use the “MISSION” tab in the LabPad as inspiration. Write your answers here: 4. Describe any obtained results. Explain any obtained results. Were these results expected or unexpected? Hint: You can use the “MEDIA” tab in the Lab Pad to find relevant images from the simulation. You can also take screenshots while you are playing the simulation. Write your answers here: 5. Discuss the conclusions and implications. How do your results relate back to the original purpose and your hypothesis? Were there any systematic sources of error that could have affected the results? What did you learn? What is the importance of these findings and how can you apply them to other real-world situations? Write your answers here: 1 Copyright Labster ApS 2021 All Rights Reserved Lab Report Instructions: The lab report will be completed based on the Synthetic Biology Lab. The rough draft is worth 50 points and the final report is worth another 50 points. Please see the assignment for the rubric. Instructions for each section and the entire report can be found here: Laboratory Report Instructions - Online Writing Lab - Reed College. For this specific report, you will want to use the theory page from Labster (see figure below) to help you with your introduction and discussion. · You may also use other primary and secondary sources- just be sure to cite them. · This isn’t a research paper (we don’t want 10 pages...) but use the lab report from Labster as a guide. There isn’t a minimum per se for the draft- but this could change for the final. · Only use word and/or a pdf (we recommend saving a pdf version as tables and figures have been known to move around during submission). You may use google docs as well…just be careful with the formatting. · For documentation- you may take screenshots (like that above), and you may create any figures or tables that you feel are needed. Virtual Lab Manual Synthetic Biology Synopsis Use the specific microRNA profile of cancer cells to design an apoptotic biological circuit that is only activated in cancer cells. You are the only hope for a patient with a rare form of cancer. Learn how to use the Gateway cloning technique In the Synthetic Biology simulation, you will learn how to use the Gateway cloning technique to combine different genetic modules in an expression vector. You will learn how the Gateway cloning technique can be used to efficiently combine these modules. Use electroporation to transform the bacteria with your vector of interest and select successful transformants. You will learn all about the basics of sterile lab work and bacterial selection. Extract and isolate plasmids Your next task in the lab will be to grow the transformed bacteria and extract the plasmids using a ‘miniprep’ kit. You will learn how different buffers enable you to isolate plasmid, and not genomic, DNA from the cells. You will check if the plasmid, indeed, contains your circuit, and if it is mutation-free by digesting it with restriction enzymes and by using gel electrophoresis. In the end you will test your circuit in living cells to see if the cancer cells are the only cells that die. Will you be able to find a cure for this rare form of cancer? Learning Objectives At the end of this simulation, you will be able to… · Engineer natural systems to perform specific functions · Describe the fundamentals of the Gateway cloning technique and design your own biological circuit · Explain and perform bacterial transformation, antibiotic selection and plasmid purification · Explain and perform a restriction digest of your cloning product Techniques in Lab · Gateway cloning technique · Electroporation · Antibiotics selection · Sterile technique · Plasmid isolation with purification columns · Restriction digest · Gel electrophoresis Theory Biological circuits Biological circuits consist of biological parts that perform logical functions, analogous to an electronic circuit. Biological circuit engineering builds upon the basic mechanics of gene expression and regulation. The enormous complexity of biological systems remains a huge challenge that often leads to unpredictable outcomes. Biological circuit parts are recombinant DNA sequences, which by themselves are simply a piece of code without a function. If circuit parts are transformed into a host organism, this code will be translated into a specific protein with a specific function. Biological circuits are assembled using molecular cloning techniques. Recent research has developed standardized techniques to build circuit libraries that can be efficiently recombined to create novel circuits. Molecular cloning Molecular cloning refers to the assembly of DNA molecules into a vector and the subsequent transformation of an organism (often bacteria or yeast). Molecular cloning methods are central to biology and medicine. The term molecular cloning comprises many different techinques. A typical molecular cloning flow looks as follows: 1. Isolation of the gene of interest 2. Cloning the gene into a vector 3. Transforming the host organism with the vector construct 4. Antibiotic selection of transformed cells 5. Isolation of clones with the same genetic background Confirmation of plasmid assembly can be determined by performing DNA sequencing. It is important to confirm that the inserts have been successfully ligated into a plasmid vector with the correct conformation and reading frame. Frame shift can cause nonsense or misense mutations that lead to the expression of nonfunctional proteins. Gateway cloning The Gateway technology provides a fast and efficient route for cloning. This technology relies on the use of modified versions of the recombinases from the bacteriophage lambda. This virus inserts its genome into the host DNA using these enzymes. The Gateway cloning system uses them to achieve high efficiency, site specific recombination. The second useful characteristic of this system is its recognition sites for the clonase enzymes called att sites, and the use of different types of vectors. BP and LR reactions BP and LR reactions are the way different DNA segments are moved from one place to another within the constructs. During the BP reaction, attB and attP sites are recombined. This reaction swaps the DNA between strands, creating an attL and an attR site. During the LR reaction, attL and attR sites are similarly recombined, yielding attB and attP sites. There is a limited number of att sites, annotated with numbers. This means that there is a limited number of combinations. Each site only recombines within the group: for example, attL1 sites will only recombine with attR1 sites, yielding attB1 and attP1 sites. The orientation of these sites is also specific, and the gene construct highly predictable. The desired construct can be built by selecting for the proper att ends, the reactions, and the vectors. Vector types The recyclable att sites are ideally suited for creating vector libraries that can be used to efficiently combine different circuit parts. • Expression vector (AmpR): The expression clone is the final product of a gateway reaction. It is the complete plasmid, ready for transformation. • Donor vector (ccdB, KanR): The donor vector provides the backbone for the creation of entry clones. Donor vectors are typically denoted with names starting with pDONR. These plasmids carry attB or attP sites flanking the ccdB gene, as well as the kanamycin resistance gene. • Entry vector (KanR): The entry vectors are created from the donor vector and a DNA sequence flanked with the matching att sites. These sequences of interest are usually produced by PCR, with primers that contain the att sites. Entry vectors contain attL or attR sites flanking the sequence of interest, as well as the kanamycin resistance gene. The entry vectors are ideally suited for a library of circuit parts. • Destination Vector (ccdB, AmpR): The destination vector provides the backbone for expression clones. Destination vectors are typically denoted with names starting with pDEST. These plasmids contain the ccdB gene flanked by attL or attR sites, as well as an ampicillin resistance gene. Destination vectors also contain the origins of replications for specific hosts and additional DNA motifs. Selection The selection of the vectors of interest in the different steps of the Gateway cloning procedure is very important. For selection, the Gateway system relies on two antibiotic resistances and the ccdB gene for positive selection. The antibiotic resistance enables transformed cells to grow on a medium containing antibiotics that kill untransformed cells. ccdB encodes a bacteriotoxin that prevents DNA replication. All of the backbones for Gateway clones (pDEST and pDONR) carry this gene, and only bacteria carrying an additional ccdA gene will grow when transformed with these plasmids. Antibiotic selection The number of transformed cells is usually very low compared to the cells that did not take up the vector. Hence, the transformed cells have to be selected somehow. The most common method is based on antibiotic resistance genes that are part of the vector construct. After transformation, the cells can be grown on a rich medium containing the matching antibiotics. In this selective media, only cells containing the plasmid vector are able to multiply and produce colonies. Antibiotic selection is an important step of molecular cloning. Plasmid miniprep There are several different methods to purify plasmid DNA from bacterial cells. Miniprep is a rapid, small-scale isolation method, which relies on alkaline lysis of the cells, followed by silica column purification of the DNA. The following steps have to be performed to purify plasmids from a bacteria culture: 1. Pelleting the cells by centrifuging them at 10K rpm for 3 min. 2. Homogenizing the cells by adding a homogenization buffer and pipetting repeatedly. 3. Lysing the cells with a lysis buffer (containing a detergent) and by inverting the tube repeatedly. 4. Lysing the cells for a maximum 5 minutes at room temperature. 5. Stopping the reaction by adding a neutralization buffer, and again inverting the tube several times. White clumps appear, which are the cells’ debris. 6. Pelleting the debris by centrifuging for 10 min at 13K rpm. In the end, your plasmid will be in the supernatant (the liquid phase). 7. Applying the supernatant to the silica column and centrifuging at 13K for 1 min. Your plasmid will bind to the filter at the bottom of the column. Discard the flow-through. 8. Washing your DNA twice with a wash buffer (add, centrifuge, discard). 9. Centrifuging 1 minute without adding anything, in order to get rid of any residual buffer. 10. Transfer your column to a clean 1.5ml tube and add an elution buffer (water with 10mM Tris_HCl). Leave it on your bench for a couple of minutes before centrifuging again. 11. Checking DNA concentrations in the nano-drop. Restriction enzymes Restriction enzymes cleave the sugar-phosphate backbone of double-stranded DNA. They recognize a specific site of double-stranded DNA and cleave it within, or adjacent to, their recognition site. Restriction enzymes are a very important tool in molecular biology. They allow us to cut DNA strands in a highly predictable manner. The resulting ends are divided into: · Sticky ends: One strand is longer than the other, resulting in either a 3' or 5' overhang. · Blunt ends: Both strands are cut at the same base pair, resulting in an end without an overhang. Following are two examples of restriction enzymes XbaI (pronounced Xba-one): produces sticky ends. It recognizes the following restriction site: 5'---T CTAGA---3' 3'---AGATC T---5' I-SceI (pronounced Sce-one): has a very unique restriction site that does not naturally occur in mice or human genomes. Hence, it is very useful for highly specific restrictions. SceI recognizes the following restriction site: 5'...TAGGGATAA CAGGGTAAT...3' 3'...ATCCC TATTGTCCCATTA...5' Gel electrophoresis Gel electrophoresis is a method to separate charged macromolecules (DNA, RNA, or proteins) of different sizes and to estimate their length. Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, this technique is often used to separate DNA or RNA molecules. This is necessary, for example, in the case of DNA profiling or to study RNA integrity. Gel electrophoresis is often used to separate PCR amplified DNA fragments. The process is also useful to isolate and extract DNA fragments of a specific size. In the virtual lab we use the E-gel machine to perform gel electrophoresis (see image below). 1 Copyright Labster ApS 2020 All Rights Reserved