Unknown Insert Report

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University of Illinois, Urbana Champaign *

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251

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Biology

Date

Feb 20, 2024

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pdf

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Alina Lukose MCB 251 11/28/23 Unknown Insert Report Purpose The purpose of this experiment was to use two different modes of analysis and determine the identity of an unknown insert (Cat or Kan). Throughout this course we learned many forms of analysis such as miniprep, PCR, gel electrophoresis, transformation, etc. to identify an unknown insert. This lab allowed us to apply the knowledge that we have retained from the semester and make discoveries. Procedures In this experiment, the modes of analysis my partner and I decided to use were PCR/gel electrophoresis and ligation/transformation and plating. For ligation/transformation with plating, we test to see if there is resistance on a plate with the antibody of interest. With PCR/gel electrophoresis, we create millions of copies of our target DNA and use gel electrophoresis to separate and analyze the fragments. Ligation/Transformation with Plating: DNA Vector Isolation: 1. Get a 3 mL culture 2. Load 1.5 mL of bacterial culture to an Eppendorf tube 3. Spin in a microcentrifuge for 60s at full speed 4. Discard Supernatant 5. Load 1.5 mL of bacterial culture to the same Eppendorf tube 6. Spin in a microcentrifuge for 60s at full speed 7. Discard Supernatant 8. Add 250 mcL of P1 buffer to the pellet formed in the tube and resuspend the pellet in the buffer 9. Add 250 mcL of P2 buffer to the cell suspension

10. Invert tube 4-6 times 11. Wait for 4 mins 12. Add 350 mcL of N3 buffer to the cell lysate 13. Invert tube 4-6 times 14. Spin in a microcentrifuge for 10 mins at full speed 15. Load 750 mcL of supernatant to the Qiagen column 16. Spin in a microcentrifuge for 60s at full speed 17. Discard effluent 18. Load 500 mcL of PB buffer 19. Spin in a microcentrifuge for 60s at full speed 20. Discard effluent 21. Load 750 mcL of PE buffer 22. Spin in a microcentrifuge for 60s at full speed 23. Discard effluent 24. Dry spin in a microcentrifuge for 30s at full speed 25. Move the spin column to a new Eppendorf tube 26. Add 20 mcL of EB buffer 27. Wait 1 min 28. Spin in a microcentrifuge for 30s at full speed Ligation: - From the calculations we will add 14.35 mcL of vector plasmid, 1.66 mcL of water, and 0.988 of insert to an Eppendorf tube with 2 mcL of ligase buffer and 1 mcL of DNA ligase - Mix wit pipet - Incubate overnight for insert 1 ligated to plasmid

Transformation: 1. Attain one tube of DH5 alpha cells (170 mcL of competent cells) 2. Label two microfuge tubes with “P” and “NP” for plasmid and no plasmid, and add 80 mcL of competent cells to each tube 3. Add 4 mcL of the plasmid solution from the ligation to the “P” tube. Add nothing to the “NP” tube. 4. Incubate on ice for 30 minutes. 5. Place both tubes in the floating microfuge tube rack in a 42 degrees C water bath for 90 seconds. 6. Place both tubes on ice for 2 minutes. 7. Add 400 mcL of SOC broth to each microfuge tube. 8. Put both tubes in the microfuge rack to be taken by the prep staff to the shaker/incubator, which will grow the cells at 37 degrees C for 1 hour. 9. Receive the cells from prep staff. 10. Take 2 plates of each: LA, LA/Amp/IPTG/Xgal, La/Kan, and LA/Chl plates. 11. Spread plates with 100 mcL of culture used on each plate. Make sure to spread across the whole plate and to sterilize the spreader each time. 12. Incubate at 37 degrees overnight and stored in the refrigerator

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PCR/Gel Electrophoresis Broth Test: 1. Get a single colony from the plate with the most growth 2. Obtain a test tube with LB broth and antibiotic that was used in the plate with the growth (LA/Chl plate) 3. Dip the colony from the plate and mix 4. Incubate and grow the culture at 37 degrees C Plasmid Purification: 1. Load 1.5 mL of this bacterial culture to an Eppendorf tube 2. Spin in microcentrifuge at full speed for 60 s 3. Discard supernatant 4. Load 1.5 mL bacterial culture to the same Eppendorf tube 5. Spin for 60 s 6. Discard supernatant 7. Add 250 mcL P1 buffer to pellet 8. Add 250 mcL P2 buffer to the same Eppendorf tube 9. Invert gently 4-6 times 10. Wait for 4 mins 11. Add 350 mcL of N3 buffer to tube 12. Gently invert 4-6 times 13. Spin in the microcentrifuge for 10 mins 14. Load 750 mcL supernatant to Qiagen column 15. Spin in microcentrifuge for 60 s 16. Discard effluent 17. Load 500 mcL of PB buffer 18. Spin in microcentrifuge for 60 s 19. Discard effluent 20. Load 750 mcL of PE buffer 21. Spin in microcentrifuge for 60 s

22. Discard effluent 23. Dry spin the column in microcentrifuge for 30 s 24. Move spin column to new Eppendorf tube and add 50 mcL of EB buffer 25. Wait 1 min 26. Spin the Eppendorf tube in microcentrifuge for 30 s Polymerase Chain Reaction (PCR): 1. Transfer 2.5 mcL of the plasmid from purification to new Eppendorf tube 2. Add 12.5 mcL of 2X Paq5000 Master mix, 5 mcL of upstream primer, and 5 mcL downstream primer 3. Mix with pipet and centrifuge for 30 s 5. Take 25 mcL of culture and place into PCR tube 6. Let PCR run for 2.5 hours Gel Electrophoresis: 1. Mix 5 mcL of loading dye with 10 mcL PCR content 2. Load the mixture into the agar gel 3. Run gel at 100 V for 45 minutes 4. Place the gel under the UV light box and analyze Results - Using an online measuring tool, we were able to make a semilog graph with distances from the band lengths (distance about 65 mm)

Conclusion: - In this experiment, my partner and I had insert 1 - From the first mode of analysis (ligation/transformation), we conducted ligation and transformation for a blue-white screen. Plasmid pBLU was ligated with unknown insert 1 and transformed into bacteria, which were then plated on LA, LA/Ampicillin, LA/Kanamycin, and LA/Chloramphenicol agar plates. Growth on the LA and LA/Chloramphenicol plates, marked by white colonies, indicated successful transformation and resistance to chloramphenicol. The white colonies suggested that Beta-galactosidase did not cleave X-gal, pointing to lacZ interruption by the unknown insert. This analysis suggests that the cat gene, conferring chloramphenicol resistance, is likely the identity of unknown insert 1. - In the second mode of analysis, we utilized PCR and gel electrophoresis to determine the size of the unknown insert. After confirming bacterial growth in chloramphenicol antibiotic broth through a broth test, we isolated the ligated plasmid with unknown insert 1 during plasmid purification. PCR amplification and gel electrophoresis revealed several bright bands, with the most prominent one just above 1000 bp. Further analysis, including a semi-log graph, estimated the band size at around 1047 bp. This size corresponds closely to the expected size of the cat insert fragment (about 1015 bp) and significantly differs from the expected size of the kan insert gene (1479 bp). Therefore, based on this analysis, it can be concluded that unknown insert 1 is the cat insert gene.

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