PCR results, DNA gel images, MEGA alignments and phylogenies

Projects #1 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 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). 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 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. 1 Research Proposal 1A Introduction/ Background Maggenti (2020) states that nematodes are among the large groups of the metazoans family. Roughly less than 4% of nematode organisms are scientifically known, with worldwide species richness of approximately 106 and 108. Most of the nematode species are parasitic and are harmful to the health of plants and animals globally. For instance, the World Health Organization (WHO) released a report which revealed that global infections because of soiltransmitted nematodes account for the human disease problem of 3.8 million years lost because of disabilities (Maggenti, 2020). It is important to work with nematodes because they enhance soil quality by mineralizing nutrients into plant-available forms, controlling the population of soil organisms, consuming disease-causing organisms, and offering a food source for them. The transformations in DNA sequencing approaches have seen precise and rapid identification of many organisms like nematodes. Scientific scholars have attempted to address the genetic taxonomy of nematodes with the help of the 18S rRNA. Worm lysis and the 18S gene are commonly studied topics in biology. Abbreviated as 18 rRNA, 18ribosomal RNA forms part of the ribosomal RNA. 18S rRNA is a eukaryotic cytosolic 16S ribosomal RNA homolog in plastids and prokaryotes. 18S rRNA is among the mostly used genes in phylogenetic studies. It is also a vital marker in environmental biodiversity screening (Borges et al., 2022). Precisely, it is an important marker by randomly targeting polymerase chain reaction (PCR). rRNA gene sequences are easily accessible because the highly flanking regions make it easier to use universal primers. The gene is recognized for reconstructing the 2 metazoan tree of life. It also contributed to the most existing revolutionary change in conceptualizing metazoan relationships. Research Question/ Problem/ Hypothesis Statement The primary research problem that this proposal intends to solve is determining the 18S RNA gene role in reconstructing vigorous phylogenetic trees. The study also focuses on the significance of worm lysis in genetics. The research questions are listed below: RQ1: What is the 18S RNA gene’s role in reconstructing vigorous phylogenetic trees? RQ2: What is the significance of worm lysis in genetics? This proposal is important because it will add knowledge regarding the 18S RNA gene’s role in reconstructing vigorous phylogenetic trees and the significance of worm lysis in genetics. Methods The study will perform a qualitative systematic review method to identify information about the studied topic from peer-reviewed sources (Williams et al., 2021). The method will uncover a new understanding of the subject. Results Borges et al. (2022) concluded that the 18S RNA gene is vital in reconstructing vigorous phylogenetic trees. On the other hand, Borges et al. (2022) concluded by citing that worm lysis is important in genetic mapping. 3 References Borges et al. (2021). 18S rRNA gene sequence-structure phylogeny of the Trypanosomatida (Kinetoplastea, Euglenozoa) with special reference to Trypanosoma. European Journal of Protistology, 81, 125824. https://doi.org/10.1016/j.ejop.2021.125824 Maggenti, A. R. (2020). General nematode morphology. In Manual of agricultural nematology (pp. 3-46). CRC Press. Williams et al. (2021). Re-examining systematic literature review in management research: Additional benefits and execution protocols. European Management Journal, 39(4), 521-533. Methods Methods Worm Lysis Biology scholars can perform worm lysis on a single worm or numerous worms. A biology scholar can perform this process on a pool of worms (50-60). The consideration for worm lysis is that it breaks the cell worms and identifies the nucleus (Maggenti, 2020). The researchers working as a team for this research report inserted 20 worms into the PCR tube, followed by 33uL worm lysis. Later, he put two tubes in a freezer of -800, one with the worms and the other with no worms. The tube without worms was a negative control tube to utilize in the PCR reaction. The researchers used the new kit to perform worm lysis and DNA extraction to break the cell and access the nucleus by using the two tubes, one for the regular with worms and the other for the control. The regular tube contained 1-16 worms, 2ul extraction, 0.5ul tissue, and 2ul neutralization. PCR PCR is an acronym for polymerase chain reaction (PCR) and is a three-step process needed for any DNA synthesis reaction. These steps are denaturing the template into single strands, annealing primers to specific original strands for the new strand synthesis, and stretching the new DNA strands from the primers (Mubarak et al., 2020). The researchers performed PCR with the help of DreamTaq to determine the reliability of the method. They added 1.0ul 10uM SSU26R primer, 1.0ul 10uM SSU18R primer, 25ul DreamTaq PCR master mix, and 18ul dH20 in both tubes, one for negative control and the other with worms. Gel Electrophoresis and Gel Extraction It is a laboratory procedure to separate mixtures of proteins, DNA, or RNA depending on the molecular size. The procedure ensures that the electrical field pushes the molecules to be separated through gel with tiny pores (Green & Sambrook, 2019). Therefore, the researchers run the gel to determine the reliability of the PCR, where being able to observe the bands implicates the effectiveness of the gel. The researchers first made Gels by adding 50mL 1X TAE and 0.4g of agarose to 125mL Erlenmeyer flask and heating the contents in the microwave for 60 seconds at the interval of two in thirty-second increments. After that, they allowed the mixture to cool to hold the flask comfortably for about 10 seconds. After that, they added 2uL gel green into the flask and swirled the flask to mix the contents, build and set up the trays, and drain the contents into the prepared gel mold, allowing them to solidify. The researchers loaded Gels after making them by following a specific procedure. They prepared samples for loading while allowing the gel to cool and harden. They also prepared where to load the DNA ladder and each sample. Secondly, they placed the gel mold into the gel box, loosened the plastic screws, and lowered the mold sides. They added 1X TAE to the gel box until it reached a level 1cm higher than the top gel face. They added a 24uL sample to the intended wells and a 10uL DNA ladder and used electrodes to attach the lid. After that, they run the sample at 100mA or 100mv for roughly 40 minutes. Following this process suggests the possibility of easily observing 25 to 100ng of the DNA. DNA Sequencing It is the general laboratory method for determining the precise sequence of bases or nucleotides in a DNA molecule. DNA sequencing can be achieved through different methods like synthesis and single-molecule DNA sequencing (Green & Sambrook, 2019). Like the former procedures, the researchers performed worm lysis to identify the DNA by breaking the worm cell. They also run the gel thoroughly to determine the usefulness of the PCR. They performed the procedure of running the gels by making them and loading them for about 10-20 minutes to determine the band size. It will be easier for them to see 25-100ng of the DNA with fewer constraints. References Green, M. R., & Sambrook, J. (2019). Analysis of DNA by agarose gel electrophoresis. Cold Spring Harbor Protocols, 2019(1), pdb-top100388. Maggenti, A. R. (2020). General nematode morphology. In Manual of agricultural nematology (pp. 3-46). CRC Press. Mubarak et al. (2020). An optimization and common troubleshooting solving in polymerase chain reaction technique. Systematic Reviews in Pharmacy, 11(2), 427-436. BIO-430-01 Lab Notebook Fall-2022 TABLE OF CONTENTS Page# Lab Introduction (08/22/2022)……………………………………………………………………………… 1 Worm Lysis (08/29/2022)…………………………………………………………………………………….. 1 PCR (09/02/2022)……………………………………………………………………………………………….. 1-2 Gel Electrophoresis (09/09/2022)………………………………………………………………………….. 2-3 New Worm lysis Protocol (09/12/2022)…………………………………………………………………. 3-4 Wom Lysis(09/16/2022)………………………………………………………………………………………. 4 PCR Using DreamTaq(09/19/2022)……………………………………………………………………….. 5 Absent (09/23/2022)…………………………………………………………………………………………….. 5 Running Gel Using DreamTaq(09/26/2022)……………………………………………………………. 5-7 Doing PCR on BIO-121 class worm strains (09/30/2022)…………………………………………. 7-8 Running Gel (10/03/2022)…………………………………………………………………………………….. 8-10 Worm Lysis (10/07/2022)……………………………………………………………………………………… 11 PCR (10/14/2022)…………………………………………………………………………………………………11-12 Running Gel(10/17/2022)…………………………………………………………………………………….. 12-13 Running Gel(10/21/2022)…………………………………………………………………………………….. 13-14 Mega11 (10/24/2022)…………………………………………………………………………………………….. 14 Mega11 (10/28/2022)…………………………………………………………………………………………….. 14 Mega11 (10/31/2022)…………………………………………………………………………………………….. 14 Mega11 (11/04/2022)…………………………………………………………………………………………….. 15 Mega11 (11/07/2022)…………………………………………………………………………………………… 15-16 Phylogeny Trees In Mega11 (11/11/2022)……………………………………………………………….. 16 Absent (11/14/2022)…………………………………………………………………………………………….. 16 1 Getting PCR Working ● 08/22/2022 ○ Introduction ○ Syllabus ○ Worked on tidying up the lab ○ Selected projects for the semester ○ Selected project partners ○ Worked on picking up 5 worms into a new NGM plate ● 08/26/2022 ○ Presented on what our group will be doing this semester ○ Checked up on our worms ○ Some of the worms were dead ○ Prepared our boxes for the year ● 08/29/2022 ○ Worked on worm lysis ■ The lysis will help break the cell worms and find the nucleus ○ Added 33 uL worm lysis buffer into PCR tube; picked (~20) worms into tube. From an unseeded plate and with minimal bacteria. ○ Negative control tube to use in PCR reaction (no worms) ○ Put in a freezer -80 ℃ (four days) ● 09/02/2022 ○ Discussion on two articles ○ Prepared Ice bucket ○ Took out the tubes from freezer ○ Added 2 uL PK per tube 2 ○ Place the tubes in heat block at 65℃ for 1 hour and 15 minutes ○ Planned PCR ingredients ○ Get five tubes for master mix and add ■ 81 ul dH2O ■ 112.5 ul DreamTaq PCR master mix ■ 4.5 ul 10 uM SSU18A primer ■ 4.5 ul 10 uM SSU26R primer ■ In total we will have 202.5 ul master mix ■ Then everyone gets four PCR tubes and adds 45 ul of the master mix into each of their PCR tubes. ○ To each sample add 5 ul of lysed worm mixture ● 09/09/2022 ○ Going over plans for next few weeks ○ Goal for today is to run the gel successfully ○ I’m predicting that the running of gels for PCR will be successful ○ Preparing 3 gels for PCR ■ Measure 0.40 g of agarose and transfer it to 125 mL flask x3 ■ Add 50 mL of 1X TAE ■ Microwave about 60 seconds to dissolve the mixture. If it’s not dissolved, microwave again for 30-40 seconds. If it’s not dissolved, microwave again for 10-15 seconds. ■ Once the mixture is dissolved, let the mixture cool. ■ Add 1 uL of gel red (or 2 uL of gelGreen), swirl it to mix, and then pour into the prepared. ○ We used gelGreen ○ Don’t over tighten. ■ Allow the gel to solidify for 15-20 minutes. It will appear on opaque, slightly gray/purple color. ○ Loading Gel ■ While the gel is cooling and hardening, prepare your samples for loading onto the gel. Also plan where to load each sample and the DNA ladder. ○ Thaw the sample, mix by tapping, spin down. ○ Aliquot 20 uL of your PCR samples into new. ○ Mix by tapping, and then spin down. ■ Place the gel mold into the gel box. Loosen the plastic screws and lower the mold sides. ■ Add 1X TAE to the gel box so that it is about 1 cm higher than the top face of the gel. ■ Add 24 uL of sample to the intended wells, and 10 uL of DNA ladder. ■ Attach the lid with electrodes. ■ Attach electrodes to the power supply. 3 ■ Run the sample at 100 mv or 100 mA for 30-40 min. Visualize the UV light box (for gel red). GelGreen can be visualized by either UV or LED light. (UV damages the DNA so LED is better. However, UV is probably more sensitive at detecting faint bands. ■ To estimate the band size you might need to run the gel for 10-20 minutes long. ■ You should see 25-100 ng of DNA easily. ○ For single clear bands of the correct size: ■ Cut it using a gel extraction tip on a p1000 micropipette and expel the gel into a labeled 1.5 uL tube. ■ If there is a band in your negative control, or no bands consult your instructor. ■ If two or more bands in one lane, you may want to try to increase annealing temperature. ○ My gel didn’t work. (No bands visible). So, no picture. ○ Drawing: ● 09/12/2022 ○ Going over plans for the day ○ Group 1 & 2 help with plates from 121 and plan new protocol ● New protocol: ○ DNA extractions of a few individuals. 4 ○ Useful for genotyping crosses. ○ All steps are done at room temperature. ○ First start a thermocycler program to heat to 55℃ for 10 minutes then 95℃ for three minutes. ○ Aliquot 2.0 uL extraction in a PCR tube. ○ Add 0.5 uL tissue preparation solution to it. ○ Mix the solution by pipetting. ○ Pick 16 gravid adult animals into the solution in a PCR tube. ○ Centrifuge the tubes (2-3 seconds, ≤ 6000 rpm/2,000xg). ○ Put the tube in the thermocycler and continue the program. ○ When it is complete, centrifuge the tubes briefly. ○ Add 2.0 uL neutralization solution. ○ Mix by pipetting. ○ The extract can either be stored at 4℃ for future or it can be used for PCR. ○ Samples will be ready for PCR within 15 minutes. ○ We found that SapphireAmp Fast PCR Master Mix further reduces total time and works more reliably than the PCR Reaction Mix provided in the Extract-N-Amp XNAT2 kit. ● ● ● ● ● 09/16/2022 Worm lysis Purpose: To perform DNA extraction and worm lysis using the new kit Expected result: Break the cell and get to the nucleus Procedure: ○ Each person gets two PCR tube ■ One for the control and one for the regular with worms ○ In the regular tube: ■ 2 ul of extraction ■ 0.5 ul of tissue ■ 1-16 worms ■ Then put in the thermocycler ● 55C (for 10 minutes) ● 95C (for 3 minutes) ■ 2 ul of neutralization ■ Then half of us do the dream taq and half do the new protocol ● New protocol ○ 5.2 ul of water ○ 10 ul of extraction N AMP ○ 0.4 ul of 18s ○ 0.4 ul of 26 r ○ 4 ul of worm extract ■ In total we have 20 ul ■ No need for master mix 5 ■ Put back in thermocycler and run the PCR overnight ○ Protocol link: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documen ts/249/244/xnat2bul.pdf ● ● ● ● ● 09/19/2022 PCR using DreamTaq Purpose: Perform PCR using the DreamTaq method Expected results: PCR will work with DreamTaq method Procedure: ○ Get two tubes per person ○ To these tubes add: ■ 18 ul dH2O ■ 25 ul DreamTaq PCR master mix ■ 1.0 ul 10 uM SSU18A primer ■ 1.0 ul 10 uM SSU26R primer ■ In total we have 45.0 ul sample in each tube ■ Add 4.5 ul of the worm lysis into each tube ■ Label the tubes: 1st tube has worms and 2nd tube is the negative control ○ Put the tubes in the thermocycler for 2.5 hours ○ The cycle of PCR in the machine are as follows: ■ 3:00 at 94 C to denature ■ 0:30 at 92 C ■ 0:30 anneal at 55 C ■ 1:00 extension at 72 C (1 minute for extension for every 1000 bp of sequence) ■ Go to 2 33X ■ 10:00 extension at 72 C ■ 4 C forever ■ End ■ Place in -20 C freezer (stopping point) ● 09/23/2022 ● Sick, didn’t came to lab ● 09/26/2022 ● Running the gel 6 ● Purpose: Run the gel properly to see if PCR works. ● Expected results: To see the bands ● Procedure: ○ Make Gels: ■ Add 0.4 g of agarose to 125 mL erlenmeyer flask ■ Add 50 mL 1X TAE ■ Heat in microwave for 60 seconds in 2 30-second increments ■ Let the mixture cool until you can hold the part of the flask comfortably for 10 seconds ■ Build the trays ■ Add 2 uL of gel green into the flasks and swirl the flask to mix ■ Set up the trays (sides up, plastic screws secure, and comb in place) ■ Pour the mixture into the prepared gel mold ■ Let the gel sit for 15-20 minutes to solidify ○ Loading Gels ■ While the gel is cooling and hardening, prepare your samples for loading onto the gel. Also plan where to load each sample and the DNA ladder. ■ Place the gel mold into the gel box. Loosen the plastic screws and lower the mold sides. ■ Add 1X TAE to the gel box so that it is about 1 cm higher than the top face of the gel. ■ Add 24 uL of sample to the intended wells, and 10 uL of DNA ladder. ■ Attach the lid with electrodes. ■ Attach electrodes to the power supply. ■ Run the sample at 100 mv or 100 mA for 30-40 min. Visualize the UV light box (for gel red). GelGreen can be visualized by either UV or LED light. (UV damages the DNA so LED is better. However, UV is probably more sensitive at detecting faint bands. ■ To estimate the band size you might need to run the gel for 10-20 minutes long. ■ You should see 25-100 ng of DNA easily. ○ For single clear bands of the correct size: ■ Cut it using a gel extraction tip on a p1000 micropipette and expel the gel into a labeled 1.5 uL tube. ■ If there is a band in your negative control, or no bands consult your instructor. ■ If two or more bands in one lane, you may want to try to increase annealing temperature. ■ My PCR worked. ■ Image: 7 ■ ● ● ● ● ● 09/30/2022 Doing PCR Purpose: To conduct PCR on the bio-121 class worm strains Expected results: PCR will work with the bio 121 class worm strains Procedure: ○ DNA extractions of a few individuals. ○ Useful for genotyping crosses. ○ All steps are done at room temperature. ○ First start a thermocycler program to heat to 55℃ for 10 minutes then 95℃ for three minutes. ○ Use 4 PCR tubes ○ Number the tubes from 1 to 4 ○ Aliquot 2.0 uL extraction in a PCR tube. ○ Add 0.5 uL tissue preparation solution to it. ○ Mix the solution by pipetting. ○ Pick 16 gravid adult animals into the solution in a PCR tube. ○ Tube number 1,2, 3 has worms and 4th one is the negative. ○ Centrifuge the tubes (2-3 seconds, ≤ 6000 rpm/2,000xg). ○ Put the tube in the thermocycler for 13 minutes and continue the program. ○ When it is complete, centrifuge the tubes briefly. ○ Add 2.0 uL neutralization solution. 8 ○ Take four new PCR tubes and make a mixture by following below procedure ■ Add 18 uL of dH2O ■ Add 25 uL of DreamTaq PCR master mix ■ Add 1.0 uL of 10 uM SSU18A primer ■ Add 1.0 uL of 10 uM SSU26R primer ■ Then add 4.5 uL of the worm mixture and the negative DNA into these tubes ■ In total we will have 49.5 uL of PCR mix. ○ Mix by pipetting. ○ Then put them in the thermocycler for PCR ○ The extract can either be stored at 4℃ for future or it can be used for PCR. ○ Samples will be ready for PCR within 15 minutes. ● ● ● ● ● 10/03/2022 Running Gel Purpose: The purpose is to run the gel properly, to see if PCR works. Expected results: To see the bands Procedure: ○ Make Gels: ■ Add 0.4 g of agarose to 125 mL erlenmeyer flask ■ Add 50 mL 1X TAE ■ Heat in microwave for 60 seconds in 2 30-second increments ■ Let the mixture cool until you can hold the part of the flask comfortably for 10 seconds ■ Build the trays ■ Add 2 uL of gel green into the flasks and swirl the flask to mix ■ Set up the trays (sides up, plastic screws secure, and comb in place) ■ Pour the mixture into the prepared gel mold ■ Let the gel sit for 15-20 minutes to solidify ○ Loading Gels ■ While the gel is cooling and hardening, prepare your samples for loading onto the gel. Also plan where to load each sample and the DNA ladder. ■ Place the gel mold into the gel box. Loosen the plastic screws and lower the mold sides. ■ Add 1X TAE to the gel box so that it is about 1 cm higher than the top face of the gel. ■ Add 24 uL of sample to the intended wells, and 10 uL of DNA ladder. ■ Attach the lid with electrodes. ■ Attach electrodes to the power supply. ■ Run the sample at 100 mv or 100 mA for 30-40 min. Visualize the UV light box (for gel red). GelGreen can be visualized by either UV or 9 ■ ■ ■ ■ ■ ■ ■ LED light. (UV damages the DNA so LED is better. However, UV is probably more sensitive at detecting faint bands. To estimate the band size you might need to run the gel for 10-20 minutes long. You should see 25-100 ng of DNA easily. ○ For single clear bands of the correct size: Cut it using a gel extraction tip on a p1000 micropipette and expel the gel into a labeled 1.5 uL tube. If there is a band in your negative control, or no bands consult your instructor. If two or more bands in one lane, you may want to try to increase annealing temperature. My PCR amplified, but the negative control sample didn’t work (It has a band) Image: (1,2,3 samples with worm, and 4 is the negative control) 10 11 ● ● ● ● ● 10/07/2022 Worm Lysis Purpose: To break the worm cell and to get to the DNA Expected results: Being successful at getting at the DNA Procedure: ○ I used the group 11 & 12 worms (GH TM6560, KHTM6566 B, TM6566 EW A) ○ DNA extractions of a few individuals. ○ Useful for genotyping crosses. ○ All steps are done at room temperature. ○ First start a thermocycler program to heat to 55℃ for 10 minutes then 95℃ for three minutes. ○ Use 4 PCR tubes ○ Number the tubes from 1 to 4 ○ Aliquot 2.0 uL extraction in a PCR tube. ○ Add 0.5 uL tissue preparation solution to it. ○ Mix the solution by pipetting. ○ Pick 16 gravid adult animals into the solution in a PCR tube. ○ Tube number 1,2, 3 has worms and 4th one is the negative. ○ Centrifuge the tubes (2-3 seconds, ≤ 6000 rpm/2,000xg). ○ Put the tube in the thermocycler for 13 minutes and continue the program. ○ When it is complete, centrifuge the tubes briefly. ○ Add 2.0 uL neutralization solution. ○ Then put in the freezer ● ● ● ● ● 10/14/2022 PCR Purpose: Perform PCR using the DreamTaq method. Expected results: PCR will work with the DreamTaq method Procedure: ○ Get four PCR tubes ○ To these tubes add: ■ 18 ul dH2O ■ 25 ul DreamTaq PCR master mix ■ 1.0 ul 10 uM SSU18A primer ■ 1.0 ul 10 uM SSU26R primer ■ In total we have 45.0 ul sample in each tube ■ Add 4.5 ul of the worm lysis into each tube ■ Label the tubes: 1st, 2nd, and 3rd tube has worms and 4th tube is the negative control ○ Put the tubes in the thermocycler for 2.5 hours ○ The cycle of PCR in the machine are as follows: ■ 3:00 at 94 C to denature 12 ■ 0:30 at 92 C ■ 0:30 anneal at 55 C ■ 1:00 extension at 72 C (1 minute for extension for every 1000 bp of sequence) ■ Go to 2 33X ■ 10:00 extension at 72 C ■ 4 C forever ■ End ■ Place in -20 C freezer (stopping point) ● ● ● ● ● 10/17/2022 Running Gel Purpose: Run the gel properly, to see if PCR works. Expected results: To see the bands. Procedure: ○ Make Gels: ■ Add 0.4 g of agarose to 125 mL erlenmeyer flask ■ Add 50 mL 1X TAE ■ Heat in microwave for 60 seconds in 2 30-second increments ■ Let the mixture cool until you can hold the part of the flask comfortably for 10 seconds ■ Build the trays ■ Add 2 uL of gel green into the flasks and swirl the flask to mix ■ Set up the trays (sides up, plastic screws secure, and comb in place) ■ Pour the mixture into the prepared gel mold ■ Let the gel sit for 15-20 minutes to solidify ○ Loading Gels ■ While the gel is cooling and hardening, prepare your samples for loading onto the gel. Also plan where to load each sample and the DNA ladder. ■ Place the gel mold into the gel box. Loosen the plastic screws and lower the mold sides. ■ Add 1X TAE to the gel box so that it is about 1 cm higher than the top face of the gel. ■ Add 24 uL of sample to the intended wells, and 10 uL of DNA ladder. ■ Attach the lid with electrodes. ■ Attach electrodes to the power supply. ■ Run the sample at 100 mv or 100 mA for 30-40 min. Visualize the UV light box (for gel red). GelGreen can be visualized by either UV or LED light. (UV damages the DNA so LED is better. However, UV is probably more sensitive at detecting faint bands. ■ To estimate the band size you might need to run the gel for 10-20 minutes long. 13 ■ You should see 25-100 ng of DNA easily. ○ For single clear bands of the correct size: ■ Cut it using a gel extraction tip on a p1000 micropipette and expel the gel into a labeled 1.5 uL tube. ■ If there is a band in your negative control, or no bands consult your instructor. ■ If two or more bands in one lane, you may want to try to increase annealing temperature. ■ My gel didn’t work because the gel was running the opposite direction. ● ● ● ● ● 10/21/2022 Running Gel Purpose: Run the gel properly, to see if PCR works. Expected results: To see the bands. Procedure: ○ Make Gels: ■ Add 0.4 g of agarose to 125 mL erlenmeyer flask ■ Add 50 mL 1X TAE ■ Heat in microwave for 60 seconds in 2 30-second increments ■ Let the mixture cool until you can hold the part of the flask comfortably for 10 seconds ■ Build the trays ■ Add 2 uL of gel green into the flasks and swirl the flask to mix ■ Set up the trays (sides up, plastic screws secure, and comb in place) ■ Pour the mixture into the prepared gel mold ■ Let the gel sit for 15-20 minutes to solidify ○ Loading Gels ■ While the gel is cooling and hardening, prepare your samples for loading onto the gel. Also plan where to load each sample and the DNA ladder. ■ Place the gel mold into the gel box. Loosen the plastic screws and lower the mold sides. ■ Add 1X TAE to the gel box so that it is about 1 cm higher than the top face of the gel. ■ Add 24 uL of sample to the intended wells, and 10 uL of DNA ladder. ■ Attach the lid with electrodes. ■ Attach electrodes to the power supply. ■ Run the sample at 100 mv or 100 mA for 30-40 min. Visualize the UV light box (for gel red). GelGreen can be visualized by either UV or LED light. (UV damages the DNA so LED is better. However, UV is probably more sensitive at detecting faint bands. ■ To estimate the band size you might need to run the gel for 10-20 minutes long. 14 ■ You should see 25-100 ng of DNA easily. ○ For single clear bands of the correct size: ■ Cut it using a gel extraction tip on a p1000 micropipette and expel the gel into a labeled 1.5 uL tube. ■ If there is a band in your negative control, or no bands consult your instructor. ■ If two or more bands in one lane, you may want to try to increase annealing temperature. ■ My gel didn’t work because the wires were connected in the opposite directions. ● ● ● ● ● 10/24/2022 Mega11 Purpose: Learning how to use Mega11 Expected results: Be able to understand the usage of Mega11 Procedure: ○ Watching tutorial videos on how to use Mega11 ● ● ● ● ● 10/28/2022 Mega11 Purpose: Learning how to use Mega11 Expected results: Be able to understand the usage of Mega11 Procedure: ○ Read written instructions on how to use Mega11 ● ● ● ● ● 10/31/2022 Mega11 Purpose: Learning how to use Mega11 Expected results: Be able to understand the usage of Mega11 Procedure: ○ Click on Mega11 software to open ○ Then click on Alignment ○ Then choose edit/build alignment and create a new alignment ○ Then choose DNA as the type of alignment ○ A new window will open ○ In this new window click on web and choose query genbank ○ In web search the gene number and click on add alignment ○ A new window will open and it will load the sequence ○ Click on green okay button on the new window ○ The sequence will be successfully added to the Mega11 ○ To have multiple sequence follow the steps from 6 through 9 15 ● ● ● ● 11/04/2022 Mega11 Purpose: Learning How to use Mega11 and build a tree of sequences Expected results: Be able to align the sequences and make the tree of all the sequences ● Procedure: ○ Click on Mega11 software to open ○ Then click on Alignment ○ Then choose edit/build alignment and create a new alignment ○ Then choose DNA as the type of alignment ○ A new window will open ○ In this new window click on web and choose query genbank ○ In web search the gene number and click on add alignment ○ A new window will open and it will load the sequence ○ Click on green okay button on the new window ○ The sequence will be successfully added to the Mega11 ○ To have multiple sequence follow the steps from 6 through 9 ○ One you have all of the sequences in , then click on alignment and select alignment by muscle ○ Then click on the data tab and choose export alignment in the mega format and save the file. ○ Then go to the home (original) window of the mega11 ○ Click on the phylogeny tab and select a tree of your choice ( I chose the construct/test neighbor-joining tree) ○ A new window will open and in that new window select bootstrap method under the phylogeny test ○ Then click okay, to run the test. ○ The tree will be build ○ To save the session, click on file and select save current session. ● ● ● ● 11/07/2022 Mega11 Purpose: Learning How to use Mega11 and build a tree of sequences Expected results: Be able to align the sequences and make the tree of all the sequences ● Procedure: ○ Click on Mega11 software to open ○ Then click on Alignment ○ Then choose edit/build alignment and create a new alignment ○ Then choose DNA as the type of alignment ○ A new window will open ○ In this new window click on web and choose query genbank ○ In web search the gene number and click on add alignment 16 ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ ○ A new window will open and it will load the sequence Click on green okay button on the new window The sequence will be successfully added to the Mega11 To have multiple sequence follow the steps from 6 through 9 One you have all of the sequences in , then click on alignment and select alignment by muscle Then click on the data tab and choose export alignment in the mega format and save the file. Then go to the home (original) window of the mega11 Click on the phylogeny tab and select a tree of your choice ( I chose the construct/test neighbor-joining tree) A new window will open and in that new window select bootstrap method under the phylogeny test Then click okay, to run the test. The tree will be build To save the session, click on file and select save current session. ● ● ● ● 11/11/2022 Absent due to sickness, however worked on phylogeny trees in mega11 at home Phylogeny Trees In Mega11 Purpose: The purpose of today’s lab is to generate a phylogenetic tree with the given accession numbers and DNA sequences on Canvas and to verify them to the trees in the readings ● Procedure: ○ Open MEGA 11 and follow the procedure for aligning sequences in the program. ○ In the search bar on the NCBI database, paste the accession numbers Dr. Grana provided on Canvas ○ Be sure to add the sequence for the out-group. The out-group is Gordius aquaticus Accession number: X80233 ○ Building the Phylogenetic Tree ○ Once you have all 8 sequences added, select “Alignment” and then “Align by MUSCLE” ○ Click on the Data tab and choose “Export Alignment” → “MEGA Format” ○ Go back to the home window of MEGA 11 and select the Phylogeny tab. ○ Select “Construct/Test Neighbor-Joining Tree” ○ In the Analysis Preferences window select the “Bootstrap method” option from the Phylogeny Test drop-down menu. Then click ok to build the tree ○ The phylogeny tree will then be generated. ○ To save the tree, click on “File” and select “Save Current Session” ● 11/14/2022 ● Absent (sick) 17