Team:iBowu-China/Protocols

Sticky Animated Navigation Bar

Project
Parts
Interlab
Human Practices
Modeling
Judging form
Team

Table of Contents
1. Preparation of liquid LB solution for competent cell culture and plasmid extraction
2. Preparation of solid LB solution for E. Coli competent cell culture petri dishes
3. Preparation of agarose gel
4. Polymerase Chain Reaction
5. Bacterial Colony Polymerase Chain Reaction
6. Gel Electrophoresis
7. Recycling of PCR products
8. Restriction Enzyme Reaction
9. Gibson Assembly
10. Preparation of petri dishes for competent cell culture
11. E.Coli Competent Cell Culture Transfection
12. Incubation
13. Plasmid Extraction
14. Cell-Free Synthesis
Experiment Protocol

iBowu-China has compiled an account of everything that has happened in the lab over the summer. We are excited to show you how busy we have been in iGEM 2019! We believe that sharing our notes in labs will convey the passion and dedication of our teammembers, as we continuously push our limits to achieve high quality results.

1. Preparation of Liquid LB solution for competent celll culture and plasmid extraction
Ingredients 100ml 250ml
NaCl 1g 2.5g
Tryptone 1g 2.5g
Yeast 1g 2.5g
2. Preparation of solid LB solution for E. Coli competent cell culture petri dishes
Ingredients 100ml 250ml
NaCl 1g 2.5g
Tryptone 1g 2.5g
Yeast 0.5g 1.25g
Agarose 1.5g 3.75g
Kana Antibiotic 100μl 250μl
3. Preparation of Agarose Gel
  1. Fold a weighing paper
  2. Place the folded weighing paper on an electronic balance, press ‘peeling’ to zero the balance
  3. Weigh the corresponded mass of agarose into the conical flask
  4. Pour respective volume of TAE into the conical flask
  5. Heat the solution with microwave for 3 minutes
  6. Remove the conical flask from the microwave for inspection. If the agarose is completely dissolved, proceed to step 7. If the agarose is not completely dissolved, repeat step 5.
  7. After cooling, add SybrSafe®(1/10000 v/v solution of the mixture) to the solution
  8. Set up the gel mould
  9. Gently pour the gel inside the mould, making sure the liquid is evenly spread
4. Polymerase Chain Reaction
  1. Set up a PCR system (Always add the Polymerase last in case of mistake)
  2. Place in PCR machine and set the program as needed
  3. 30min before PCR ends, make agarose gel for DNA recycling
  4. Add 6x Loading Buffer into each system
  5. The first lane of the left-hand side should always be the Marker (Trans2K DNA Marker)
PCR System (50μl)
Ingredients Volume
5*Prime Star Buffer 10 μl
dNTP 4μl
Forward Primer 1.5μl
Reverse Primer 1.5μl
DNA template 1μl
Prime Star Polymerase 1μl
ddH2O 31μl
PCR Program
Pre-Denaturing 98°C, 2 min 1 cycle
Denaturing 98°C, 15s 35 cycles
Annealing 55°C, 15s 35 cycles
Extending 68°C, 1kb/min 35 cycles
Further Extending 68°C, 5 min 1 cycle
5. Bacterial Colony Polymerase Chain Reaction
  1. 30min before PCR ends, make agarose gel for DNA recycle (see step 6 for further details)
  2. Pick a single colony with gun tip and emerge it underneath the surface of the system. Blend multiple times
  3. Pipette 1μL of PCR system on the blocks already drown on the replicate plate
  4. Pipette 1μL of PCR system on the blocks already drown on the replicate plate
  5. Add corresponded primers (Primer F & Primer R), 1μL each to the tubes
  6. Centrifuge shortly with the microcentrifuge
  7. Vortex blending
  8. Place in the PCR machine and set the program as needed (See charts below for further details
6. Gel Electrophoresis
  1. Emerge the gel in the TAE buffer, making sure the liquid covers all the wells
  2. Make sure the wells are placed toward the cathode
  3. Pipette 20% volume of 6x Loading buffer into the samples, blend with vortex
  4. Always put a marker in the leftmost lane
  5. Add sample that is well mixed with loading buffer into the gel, record the order on the lab note
  6. Electrophoresis should last for 30min, 150v.
  7. If Marker appears too short, run the gel for an additional 30 minutes with close monitoring
  8. After electrophoresis, obtain an image with the gel inspection machine and smart phone
7. Recycling of PCR Products
  1. Prepare a 1.5ml tube (Marked with the amplified sequence name)
  2. Placed the excised gel fragment in a 1.5ml Eppendorf tube
  3. Compress the gel on the dip of the tube using a microcentrifuge. Estimate gel volume
  4. Pipette equal volume of Buffer PN into the tube, mix with vortex, and place the tube inside a 50 ̊C metal bath until the gel fragment is fully dissolved
  5. Balance a CA2 spin column with 500μL Buffer BL during the time of gel dissolving 8. Centrifuge the spin column for 1min at 12000rpm, discard the flow- through
  6. Apply the dissolved gel solution into the CA2 spin column, letting stand for 2min and centrifuging at 12000rpm for 1min
  7. Collect all the flow-through back to the CA2 spin column, centrifuge at 12000rpm for 1min
  8. Discard the flow-through and pipette 600μL of Buffer PW to wash the CA2 column. Let stand for 2min then centrifuge at 12000rpm for 1min, discard the flowthrough
  9. Pipette again 600μL Buffer PW, let stand for 2min, 12000rpm for 1min, and discard the flow-through
  10. Put back the CA2 spin column, centrifuge 12000rpm for 3min without adding anything to get rid of the buffer.
  11. Apply the CA2 spin column to a new 1.5ml tube (already labeled with code on the lid, plasmid name on the body) and put inside a metal bath with lid open at 50 ̊C for 5min
  12. Pipette 30μL of buffer EB to the silicon film of the CA2 spin column, let stand in a 50 ̊C metal bath with lid closed
  13. Obtain DNA concentration with a microplate reader, note it on the body of the tube(under the name of plasmid) and date of extraction underneath that. Update into the strain list
8. Restriction Enzyme Reaction
  1. Use Nanodrop to test for the concentration of the recycled PCR products
  2. Obtain 1-2 μl of target fragment
  3. Prepare the reaction system according to the table below
  4. Incubate the system at 37°C for three hours
  5. Gel Electrophoresis and recycling of the target fragment (See steps 6 and 7 for details)
Restriction Enzyme System (50μl)
Ingredients Volume
NED Enzyme I 1μl
NEB Engyme II 1μl
Target Fragment 1-2μl
10*Cutsmart Buffer 5μl
ddH2O 41-42μl
9. Gibson Assembly
  1. Using 5μL smart mix to construct a Gibson assembly system
  2. Set PCR program as: 50 ̊C 20min 12 ̊C forever
  3. Blend Gibson system with 100μL of competent cells, ice bath over 30 min (Allow thorough contact of DNA and cells, enabling DNA to enter the cell wall)
  4. Heat shock at 42 ̊C for 90s (Cells swell at high temperature, allow thorough contact of the cell membrane and cell wall, enabling DNA to pass through the cell membrane into the cytoplasm)
  5. Ice bath for 5min(Cell shrinks at low temperature, dragging DNA on the wall into the cell)
  6. Pipette 200μL of all nutrient broth into the tube(operate inside the bench), shake the bacteria in a shaker at 37 ̊C for 1h30min
  7. Spread the bacteria on plates. Observe after 16h~20h
10. Preparation of Petri Dishes for Competent Cell Culture
  1. Solid LB solutiono prepared
  2. Carefully pour the solution into clean petri dishes in ventilated Cell Culture Hood
  3. Allow the solution to dry over 30 minutes
  4. Seal the petri dishes with tape
  5. Store in 4 ̊C fridge, upside down to prevent accumulation of moisture
11. E.Coli Competent Cell Culture Transfection
  1. Place 50μl of Trans5α competent cells culture onto ice
  2. Add the reacted fragment-backbone mixture solution into the competent cell culture
  3. Place the mixed solution onto ice for 30 minutes
  4. Stimulate the solution in 42 ̊C water bath for 60 seconds
  5. Place the solution onto ice for two minutes
  6. Add 1ml of liquid LB without antibiotics in ventilated Cell-Culture Hood
  7. Proceed with step 12
12. Incubation
  1. Prepare and label an empty 15ml test tube
  2. Pipette 400μl of liquid LB solution into the test tube
  3. Pipette the bacteria solution into the test tube with liquid LB
  4. Place the test tube into 37 ̊C incubation for 45 minutes
13. Plasmid Extraction (Omega E.Z.N.A. Plasmid DNA Mini Kid I)
  1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hr at 37°C with vigorous shaking (~ 300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an endA negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a® and JM109®.
  2. Centrifuge at 10,000 x g for 1 minute at room temperature
  3. Decant or aspirate and discard the culture media
  4. Add 250 μL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields
    Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page 6.
  5. Transfer suspension into a new 1.5 mL microcentrifuge tube
  6. Add 250 μL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
    Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO2 in the air.
  7. Add 350 μL Solution III. Immediately invert several times until a flocculent white precipitate forms.
    Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation
  8. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step
  9. Prepare the vacuum manifold according to manufacturer’s instructions
  10. Connect the HiBind® DNA Mini Column to the vacuum manifold
  11. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column
  12. Turn on the vacuum source to draw the sample through the column
  13. Turn off the vacuum
  14. Add 500 μL HBC Buffer.
    Note: HBC Buffer must be diluted with 100% isopropanol before use. Please see Page 6 for instructions
  15. Turn on the vacuum source to draw the buffer through the column
  16. Turn off the vacuum
  17. Add 700 μL DNA Wash Buffer.
    Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6 for instructions
  18. Turn on the vacuum source to draw the buffer through the column
  19. Turn off the vacuum
  20. Repeat Steps 17-19 for a second DNA Wash Buffer wash step
  21. Transfer the HiBind® DNA Mini Column to a 2 mL Collection Tube
  22. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
    Note: It is important to dry the HiBind® DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications
  23. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube
  24. Add 30-100 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
    Note: The efficiency of eluting DNA from the HiBind® DNA Mini Column is dependent on pH. If using sterile deionized water, make sure that the pH is around 8.5
  25. Let sit at room temperature for 1 minute.
  26. Centrifuge at maximum speed for 1 minute.
    Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.
  27. Store DNA at -20°C
14. Cell-Free System
  1. Preparation of Cell-Free System
  2. Incubate the sample in a sharking incubator.
Cell-Free System
Reagent Amount (μl)
E.coli extract 20μl
2.5X reaction buffer 20μl
50 mM amino acids 1.25μl
75 mM Methionine 1μl
DNA template 1μl
Distill water To a final volume of 50μl