Team:LaVerne-Leos/Notebook

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Notebook

• Notebook
• Protocols

• Notebook
• Protocols

Protocols

• Minipreps
• Digestion & Ligation
• Transformations
• Gel Purification

Qiagen QIAprep Spin Mini Prep Protocol

1. 1. Pellet 1-5ml of bacterial overnight culture by centrifugation at >8000rpm for 3min at room temp (15-25°C) in a 1.7 microcentrifuge tube. Discard supernatant. Repeat as many times as necessary to pellet 1-5mL of bacteria.
2. 2. Resuspend pelleted bacterial cells in 250µl Buffer P1.
3. 3. Add 250µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times until solution becomes blue. Do not allow the lysis reaction to proceed for more than 5min. 
4. 4. Add 350µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6times. The solution will turn colorless. 
5. 5. Centrifuge for 10min at 13,000 rpm.
6. 6. Add 850µl of supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60s and discard the flow-through.
7. 7. Wash the QIAprep spin column by adding 750µl Buffer PE. Centrifuge for 60s  and discard the flow-through.
8. 8. Centrifuge for 1min to remove residual wash buffer.
9. 9. Place the QIAprep column in a clean 1.7ml microcentrifuge tube for DNA collection. To elute DNA, add 50µl Nuclease-free water to the center of the QIAprep spin column, let it stand for 1min and centrifuge for 1min.
10. 10. Analyze collected DNA concentration and purity.

NEB BioBrick Assembly Protocol

1. Digestion:
2. 1. Thaw all enzymes need and aliquot appropriate amounts.
3. 2. Pipette the amount of nuclease free water needed to make 500 ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50 uL to get the amount of water needed)
4. 3. Add appropriate amount of DNA into the nuclease free water.
5. 4. Add 3 uL of NEB Buffer to each tube.
6. 5. Add 1 uL of the first enzyme to each tube. (E for upstream, X for downstream, E for vector)
7. 6. Add 1 uL of the second enzyme to each tube. (S for upstream, P for downstream, P for vector)
8. 7. Agitate each solution to mix well.
9. 8. Incubate upstream and downstream parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.
10. 9. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.
11. Ligation:
12. 10. Thaw 10x T4 DNA ligase reaction buffer at room temperature. Mix well.
13. 11. Add 11 uL of nuclease free water into a microcentrifuge tube.
14. 12. Add 2 uL of each parts (upstream, downstream, and vector) into the tube with water.
15. 13. Add 2 uL of 10X T4 DNA Ligase Buffer into the tube.
16. 14. Add 1 uL of T4 DNA Ligase.
17. 15. Incubate at room temperature for 10 minutes.
18. 16. Incubate at 80 degrees Celsius to denature the enzymes and stop the ligation process.
19. 17. Store in a -20 degrees Celsius freezer, or transform ligated DNA.

iGEM Transformation Protocol

1. 1. Pre-chill microcentrifuge tube and thaw competent cells on ice.
2. 2. Add 50 uL of competent cells into the chilled microcentrifuge tube.
3. 3. Add 1 uL of DNA into the microcentrifuge tube.
4. 4. Incubate on ice for 30 minutes.
5. 5. Heat shock at 42 degrees Celsius for 1 minute.
6. 6. Incubate cells on ice for 5 minutes.
7. 7. Add 200 uL of SOC media into the microcentrifuge tube.
8. 8. Incubate at 37 degrees Celsius (and shake if possible) for 1-2 hours.
9. 9. Pipette 200 uL of cells onto LB plate with appropriate resistance.
10. 10. Incubate plate overnight at 37 degrees Celsius.

QIAEX II Gel Extraction Kit Protocol

1. 1. Cut DNA band from the agarose gel with a clean, sharp razor blade. Weigh a 1.7mL centrifuge tube.
2. 2. Add the DNA gel slice to weighed tube. Reweigh and subtract from original tube to find weight of DNA slice.
3. 3. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100bp; 3 volumes for 100bp-4kb; 3 volumes with 2 volumes of water for >4kb; 6 volumes when using >2% agarose gels.
4. 4. Resuspend QIAEX II by vortexing for 30s. Add QIAX II to the tube with the sample and vortex: Use 10µL of QIAEX II for ≤ 2µg DNA; 30µL for 2-10µg DNA; and an additional 30µL for each additional 10µg DNA.
5. 5. Incubate at 50°C for 10min vortexing every 2min to melt agarose and bind DNA. Keep an eye on color change. If so adjust accordingly.
6. 6. Centrifuge the sample for 30s and carefully remove supernatant.
7. 7. Wash the pellet with 500µL Buffer QX1. Resuspend by vortexing. Centrifuge for 30s and remove supernatant.
8. 8. Wash the pellet twice with 500µL Buffer PE. Resuspend pellet by vortexing. Centrifuge for 30s and remove supernatant.
9. 9. Air-dry the pellet until it becomes white. Do not vacuum dry.
10. 10. To elute DNA, add 20µL nuclease free water and resuspend the pellet by vortexing.* Incubate according to fragment size: 5min at room temp. for ≤4kb; 5min at 50°C for 4-10kb; 10min at 50°C for > 10kb.
            *Fragments larger than 10kb resuspend by flicking and inverting tube as vortexing can cause shearing.
    
11. 11. Centrifuge for 30s and pipet supernatant into clean tube. This is your purified DNA.
12. 12. Repeat steps 9 and 10 to combine eluates and increase yield.

About Us

We are the first iGEM team from University of La Verne and La Canada High School. We are an interdisciplinary team with members from biology, math, computer science, art, and library science. We are a community-engaged team that hopes to partner with other high schools, non-profits, and local businesses to create innovative solutions to global issues!

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Contact info

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  Natural Science Division Office and Biology Department
  1950 3rd St. La Verne, CA 91750
  
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  Email: ulvigemteam@gmail.com

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