Projects #1 & #2 Goal: Get PCR working
Sample Answer for Projects #1 & #2 Goal: Get PCR working Included After Question
Description
I have attached my project file in her, read that first so you can get an idea of what I’m working with. My project name is project 1. So only focus on the details of project one. Once you read it and understand it then read the literature review file and start working on it. The articles that you choose should be related to worm lysis, research in class (undergrad), finding worms. Make sure to choose more than two articles. For choosing a gene, make sure to choose 18S gene. Once the literature paper is done. Then read the file research proposal 1A rubric. Once done reading then read the directions (lab manual chapter 15) file. And follow the exact directions please and write the paper in a different word doc, the paper should be focused on the introduction and background of 18S gene amplification only and include references. You will be mentioning methods and results too but very briefly not in details. Main focus will be background and introduction. Once done with this paper then read the research proposal 1B file and follow the directions for that and write it down in a different word document. So, in total you will submit 3 word documents to me. One will be the literature review, second will be the research proposal 1A, and third will be research proposal 1B. Please read everything carefully and work on it properly. I have had tutors before who worked on it, and gave me a very bad grade. Also for the articles again I’m mentioning please make sure that they are related to my project and choose more than two articles just to be safe. Also, choose primary articles. I will put a link in here to a primary article as an example. Do not use the example article but that is just to give you an idea. Please again, make sure to follow the directions and work accordingly. I really need an A in these assignments. Please let me know if you have any questions. Thank you. Example article link: https://bmcbiol.biomedcentral.com/articles/10.1186/1741-7007-10-59
Projects #1 & #2 Goal: Get PCR working. How long will this take? Could take 3-4 weeks to entire semester, depending on how quickly sub-goal 1 is achieved. Problem: spring 2020 we could not get PCR top work and then everything shut down. PCR of 18S is critical for my Wormfinding class this fall. Groups 1 and 2 will attempt: Sub-goal 1: gather materials/make new Worm lysis buffer. Lyse and attempt PCR. Read out is correct size band on a gel. If no amplification, keep troubleshooting. Project 1: support BIOL121 Wormfinding PCR and bioinformatics this semester. Sub-goal 2 A: complete back up sequencing on all 24 or so worm strains B: adapt the protocol that works for the BIOL121 class C: trim the sequences the students and you generate so that they can be effectively BLASTed D. Explore best software for any lite bioinformatics projects in BIOL121 (web-based tree building, for example). E. Do more advanced bioinformatics using MEGA11. We need to be able to store and access sequences using something that is free, has a good interface, and is visually appealing. F. Make a folder with a set of reference sequences to work with. G. Make illustrations with references about why the 18S primer set is good (intron-exon boundary). Project 2: build a positive control for 18S rDNA amplifications. Sub-goal 2: A: once amplification is achieved, clone into the TA cloning vector. B: the vector will be purified and stored for future use C: the vector will be tested as a positive control. Determine the minimal concentration of plasmid that works. D: the bacteria containing the vector will be frozen down so that more amplification can be easily achieved in the future. Schedule The schedule given in the syllabus gives you an outline of how to proceed in the lab, but may take longer than shown. There are potential delays at every step of the project. For the noncloning project (Group 1) and we can work together to determine the series of steps you will complete together. Week Date 8/22 1 2 3 Suggested Lab Schedule Group 1: support BIOL121 Wormfinding PCR and bioinformatics this semester 1.1 1.2 1.3 Lab safety, 1.4 lab notebooks, 4.1 pipetting, 5.1 sterile technique, 3 microscopy, 2 6 worm care & picking worms 8/26 7.1 begin worm lysis, 2 6 Worm care, 7.1 finish worm lysis, 5.4 pouring plates, 5.6 day 1 materials 8/29 4.2 Master mixes, 7.5 PCR, 5.4 seeding plates, 1.6 working 9/2 7.6 Gel electrophoresis, 14 imaging a gel, 12 Serial Cloner 9/9 12 Working with sequences, 7.1 redo lysis if failed OR test lysis with a Kit! 9/12 7.5 redo PCR if failed. 9/16 7.6 redo gel electrophoresis, if failed 9/19 7.4 Hydrate primers, 7.5 set up experimental PCR 4 5 9/23 9/26 Obtain strains from BIOL121 and complete lysis. PCR with these strains. 9/30 7.7 gel extraction or PCR purification, 7.8 measure DNA quantity, 8.1 Prepare for sequencing; post-office. 10/3 Observe results, troubleshooting. 10/7 Potentially: more troubleshooting, download sequences, analyze sequences. Review 1.2 1.6. 4.3 serial dilution, 5.2 media prep, 8.2 8.3 obtaining and working with sequences 10/10 Fall Break 10/14 Prepare sequences for 121 class to use. Look into goals DEF & G from above. 10/17 You may update the Wormfinding manual, work on bioinformatics, etc. 6 7 7.6 Gel electrophoresis of PCR reaction, 7.6 gel analysis, 1.2 labeling, 14 images 8 9 10/21 10/24 10 10/28 10/31 Writing or lab work. 11/4 Writing or lab work, troubleshooting. 11 11/7 12 11/11 Writing or lab work. Build a figure about your project, or troubleshooting. 11/14 Discuss ideas for future directions. Lab work. Wrap up. 11/18 Feedback, time to work on projects/presentations. 11/21 Some work may continue. 1.5 Lab clean up, cataloging materials generated. 11/25 On Thanksgiving Break 11/28 Wrap up everything. 1.5 Lab clean up, cataloging materials generated. 12/2 Wrap up everything. 1.5 Lab clean up, cataloging materials generated. 13 14 15 Week Date Suggested Lab Schedule Group 2 8/22 1.1 1.2 1.3 Lab safety, 1.4 lab notebooks, 4.1 pipetting, 5.1 sterile technique, 3 microscopy, 2 6 worm care & picking worms 8/26 7.1 begin worm lysis, 2 6 Worm care, 7.1 finish worm lysis, 5.4 pouring plates, 5.6 day 1 materials 8/29 4.2 Master mixes, 7.5 PCR, 5.4 seeding plates, 1.6 working 9/2 7.6 Gel electrophoresis, 14 imaging a gel, 12 Serial Cloner 9/9 12 Working with sequences, 7.1 redo lysis if failed 9/12 7.5 redo PCR if failed. 9/16 7.6 redo gel electrophoresis, if failed 9/19 7.4 Hydrate primers, 7.5 set up experimental PCR 9/23 7.6 Gel electrophoresis of PCR reaction, 7.6 gel analysis, 1.2 labeling, 14 images 7.7 gel extraction or PCR purification, 7.8 measure DNA quantity, 9/26 Go over results, troubleshooting, 8.1 Prepare for sequencing; post-office. 9/30 Potentially: more troubleshooting, download sequences, analyze sequences. Review 1.2 1.6. 4.3 serial dilution, 5.2 media prep, 8.2 8.3 obtaining and working with sequences 10/3 Observe results, troubleshooting. New 5.4 Blue-white, New 9 cloning 10/7 Continue lab work. Make sure items are stored properly before break. 10/10 Fall Break 10/14 9 Ligation reactions, 5.4 pour plates for blue-white screening, 9 transformations, 10 plating. 10/17 Notice colonies that may be correct. 10/21 10 Pick colonies to grow bacteria for mini-preps Continued lab work, troubleshooting 10/24 10 Minipreps, 7.8 nanodrop, 11 digestion, 12, Serial Cloner. 10/28 8.4 Sequencing from plasmid. Continued lab work, troubleshooting 10/31 Writing or lab work. 11/4 Writing or lab work, troubleshooting. 11/7 Download sequences, analyze sequences, or troubleshooting, writing. 11/11 Writing or lab work. Build a figure about your project, or troubleshooting. 11/14 Discuss ideas for future directions. Lab work. Wrap up. 11/18 Feedback, time to work on projects/presentations. 11/21 Some work may continue. 1.5 Lab clean up, cataloging materials generated. 11/25 On Thanksgiving Break 11/28 Wrap up everything. 1.5 Lab clean up, cataloging materials generated. 12/2 Wrap up everything. 1.5 Lab clean up, cataloging materials generated. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Other things that I’d like to achieve if your groups finish early: • Imaging and processing. Nomarski and simpler techniques. • Freezing worm strains using a new protocol. (Jessica Sowa (Trehalose/DMSO) • Sending strains to the Sowa lab. Sequencing for the Sowa lab. The Sowa lab is looking for viruses that infect C. elegans. https://nematodehunters.org/. • Testing multiple primer sets and conditions. Use these on alternate strains of diverse nematodes. (these primers are in info I have from Karin Kiontke). • Try targeting a barcoding gene we have not yet examined (polymerase? See Asher Cutter). Other Options for the future or if we finish some of these projects really early: Explore using CRISPR on C. elegans genomic DNA. This would be supported through interactions with Dr. A. She is available 12-2 on M/F. (CRISPR is really appealing, but Wormfinding is my priority early on and I don’t have the capacity to also get up to speed in this field.) You could compare efficacy and check for off-target effects. There would be videos to watch and reading on your own. There is a possible 3 week module where we could do this near the end of the semester. Research Proposal 1 B Rubric – revised background 30 points The purpose of this assignment is to further develop your research proposal after getting feedback on the 1A assignment. You should be reading your literature review and other articles as you prepare the background. Eventually, the research proposal will become your research report. Followed feedback from first draft of the proposal (4 points) Introduction/background: content (17 points) • broad introduction to: your project, nematodes, benefits of working with nematodes in the classroom and/or course-based research projects, and other specifics. • Deeper introduction: o For groups 1 & 2: introduction to gene targeted by PCR, why chosen, existence in other species, potentially with a figure about the region we are amplifying. Group 1 – a lot more on nematode diversity and purpose of finding nematodes. o For group 3: introduction to nematode behavior, and behavior in general in the context of evolution, how relevant worm behavior/nervous system is to human, etc. Talk about assays to be used/readouts from the assays/analysis to be done. o For group 4: specifics on individual genes/phenotypes/RNAi. Perhaps your group could split the selections into four sets of genes so that you each detail a sub-set of the 10 or so genes we will work with (2-3 each). Include more detail on the human disease/process the gene relates to and the mechanism of how the gene is supposed to work. • Research question/problem/ or hypothesis statement along the lines of: “Cloning the 18S rDNA as a positive control for PCR is possible and useful; supporting BIOL121 will benefit students and generate data on nematode diversity; Behavior tests can be used in 121 to characterize and compare nematode species and allow introductory students to design experiments and analyze data; Genetics students will benefit from an introduction to nematodes and RNAi that has identifiable phenotypes. (students: please refine these, as its your ideas to articulate and I only wanted to give example ideas). • Ends with a statement about how this study will add knowledge to the field / benefit classes. • Figures or tables that are part of the background may also be included. Citations (4 points) • Follow the reference guidelines beginning on page 15-10 of the lab manual. • Accessed and read various articles as you developed the background section • Citations support background appropriately. • I prefer that you use original sources-peer reviewed articles and reviews, not Wormbase, though Wormbase is a good place to find relevant articles for some projects Style (5 points) • clear connections between 1) the background and proposed research and 2) the information presented in the different paragraphs (i.e. use transitions between paragraphs). • italicized gene names, species names • sentences well structured • organized presentation of material