Team:Linkoping Sweden/Experiments
Experiment protocols
DNA Kit Plate Instructions
To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3 ng of DNA in each well, following this protocol, assume that you are transforming with 200-300 pg/µL
- With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
- Pipette 10 µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
- Transform 1 µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.
- Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours. Use the resulting culture to miniprep the DNA AND make your own glycerol stock.
Protocol from iGEM wiki
Transformation - Double Heat Shock
Method
- Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
- Label 1.5 ml tubes with part name or well location.
- Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
- Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
- Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
- Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
- Close 1.5 ml tubes, incubate on ice for 3 h: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
- Heat shock tubes at 42°C for 45 sec: Timing is critical.
- Incubate on ice for 2 min: Return transformation tubes to ice bucket.
- Heat shock tubes again at 42°C for 45 sec: Timing is critical.
- Pipette 250 µL LB media to each transformation
- Incubate at 37°C for 1 hours, shaking at 170 rpm
- Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
- Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
- Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
- Count colonies for control transformation: Count colonies on the 100 μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
Protocol from iGEM wiki and iGEM Troubleshooting&Collaborations on Facebook
Transformation - Heat Shock
Method
- Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
- Label 1.5 ml tubes with part name or well location.
- Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
- Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
- Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
- Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
- Close 1.5 ml tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
- Heat shock tubes at 42°C for 45 sec: Timing is critical.
- Incubate on ice for 2 min: Return transformation tubes to ice bucket.
- Pipette 250 µL LB media to each transformation.
- Incubate at 37°C for 1 hour, shaking at 170 rpm.
- Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
- Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
- Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
Protocol from iGEM wiki
Agar with chloramphenicol
Method
- Pour 15 ml cold liquid agar in a tube. One tube equals one agar plate.
- Pipette 15 µl chloramphenicol to the same tube. If more or less agar is used take 1 µl chloramphenicol per ml agar.
- Pour the agar solution in to a petri dish. Spread it out evenly. Let dry with lids almost covering the bottom plates.
- Apply cells after directions in transformation protocol.
Heat shock competent cells, BL21 (DE3)
Method
- Take 25 ml LB medium and put it in a Eppendorf 50 ml tube.
- Pipette 50 µl cells to the LB medium. In this case BL21 (DE3).
- Incubate at 37°C, shaking at 170 rpm for 4-6 hours.
- Thaw cells on ice for 10 mins (keep cold from now on).
- Collect the cells by centrifugation for 5 mins at 1000 rfc
- Decant supernatant. Gently resuspend on 10 ml cold 0.1 M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
- Incubate on ice for 20-30 min.
- Repeat step 5, but with 2000 rfc.
- Discard supernatant. Then gently resuspend with 5 ml cold 0.1 M CaCl2 + 15% Glycerol.
- Dispense in microtubes (300 μL/tube). Freeze in -80°C.
GenElute™ Plasmid Miniprep Kit
- Centrifuge 5 ml of overnight grown competent cells for 1 min at 1200 rcf. Discard the supernatant.
- Resuspend cells. Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous.
- Lyse cells. Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. Do not vortex. Do not allow the lysis reaction to exceed 5 minutes.
- Neutralize. Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifugation at 12,000 g for 10 min.
- Prepare column. Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to miniprep column and centrifuge at 12,000 g for 1 min. Discard the flow-through liquid.
- Load cleared lysate. Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at 12,000 g for 1 min. Discard the flow-through liquid.
- Wash column. Add 700 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 g for 1 min. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 min without any additional Wash Solution to remove excess ethanol. Discard the flow-through liquid.
- Elute DNA. Transfer the column to a fresh collection tube. Add 50 µl molecular biology reagent water to the column. Centrifuge at 12,000 g for 1 min. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C.
The protocol and material used for plasmid purification was from Sigma-Aldrich
MinElute PCR Purification Kit (50)
Method
- Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. Check that the color of the mixture is yellow (similar to Buffer PB without the sample).
- Place a MinElute column in a provided 2 ml collection tube.
- Apply the sample to the MinElute column and centrifuge for 1 min. Discard flow-through and place the MinElute column back into the same collection tube.
- Add 750 µl Buffer PE to the MinElute column and centrifuge for 1 min. Discard flow-through and place the MinElute column back in the same collection tube.
- Centrifuge the column for 1 min. Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
- Place each MinElute column in a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 10 µl Buffer PB or water to the center of the MinElute membrane. (Ensure that the eluton buffer is dispensed directly onto the center of the membrane for complete elution of bound DNA.) Let the column stand for 1 min, and then centrifuge the column for 1 min.
The protocol and material used for PCR purification was from Qiagen
Gel electrophoresis with agarose
Method
- Prepare an agarose-gel: Dissolve the agarose in 100 ml 1x TAE buffer. Heat the solution in microwave to make the solution homogeneous (Be careful, the solution must not over boil).
- Cool down the solution and pour the agarose into the tank and add 1x TAE buffer until the gel is covered.
- Mix DNA samples with purple loading dye from NEB.
- Pipette the mixed samples and ladder(NEB) into the wells.
- Run at 80 V for 90 minutes.
- Carefully remove the gel from the tank and place the gel into a container filled with Ethidium Bromide and shake for 5 minutes.
- Place the gel in another container filled with 1x TAE buffer, shake for 1 min.
- Use UV light to visualize your DNA fragments.
Q5 Site-Directed Mutagenesis (New England Biolabs)
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Primers were designed using www.nebasechanger.neb.com
PCR mix:
- 12.5 μL Q5 Hot Start High-Fidelity 2X master mix
- 1.25 μL Forward/Reverse primer final conc. 0.5 μM
- 1 μL Template DNA (1-25 ng)
- 9 μL Nuclease free water
PCR conditions:
- 98 °C 30 sec
- 98 °C 10 sec (2→ 4 repeated 25 times)
- 50-72 °C 10-30 sec
- 72 °C 20-30 sec/kb
- 72 °C 2 min
KLD reaction:
- 1 μL PCR product
- 5 μL 2X KLD reaction buffer
- 1 μL 10X KLD enzyme mix
- 3 μL Nuclease free water
- Incubate 5 min
-
Mix 5 μL of the KLD reaction with 50 μL heat shock compatible cells, transformation following whichever chassis you use.
The protocol and material used for Q5 Site-Directed Mutagenesis was from New England Biolabs
QuikChange II Site-Directed Mutagenesis Kit (Agilent)
-
Primers were designed using Benchling
PCR mix:
- 5 µL 10X reaction buffer
- X µL 5–50 ng of dsDNA template
- X µL 125 ng of oligonucleotide primer #1
- X µL 125 ng of oligonucleotide primer #2
- 1 µL dNTP mix
- To 50 µL ddH2O
PCR conditions:
- 95 °C 30 sec
- 95 °C 30 sec (2→ 4 repeated 16 times)
- 55 °C 1 min
- 68 °C 1 min/kb
Method
- Add 1 µl of the Dpn I restriction enzyme (10 U/µl) directly to each amplification reaction.
- Gently and thoroughly mix each reaction mixture by pipetting the solution up and down several times. Spin down the reaction mixtures in a microcentrifuge for 1 minute and immediately incubate each reaction at 37°C for 1 hour to digest the parental (i.e., the nonmutated) supercoiled dsDNA.
- Mix 1 μL of the Dpn I reaction with 50 μL heat shock compatible cells, transformation following whichever chassis you use.
The protocol and material used for QuikChange II Site-Directed Mutagenesis Kit was from Agilent
Q5 High-Fidelity 2X Master Mix (New England Biolabs)
-
Primers were designed using Benchling
PCR mix:
- 12.5 μL Q5 High-Fidelity 2X master mix
- 1.25 μL Forward/Reverse primer final conc. 0.5 μM
- 1 μL Template DNA (1 ng-1 μg)
- 9 μL Nuclease free water
PCR conditions:
- 98 °C 30 sec
- 98 °C 10 sec (2→ 4 repeated 25-35 times)
- 50-72 °C 10-30 sec
- 72 °C 20-30 sec/kb
- 72 °C 2 min
The protocol and material used for Q5 High-Fidelity 2X Master Mix was from New England Biolabs
SDS PAGE
Method
- Grow desired product in Falcon tubes
- Induce - for every sample there should be a negative control
- Incubate in room temperature on a shaking table over night
- Check OD (to get a relative protein expression)
- Take 500 µl of sample to Eppendorf tube 2 ml
- For GroES purification: incubate at 80℃ for 40 min
- Centrifuge at 12000 g for 10 min
- For samples not incubated at 80℃: Remove supernatant and add 50 µl MilliQ-water
- Take 10 µl to a new tube
- Add 10 µl SDS sample buffer
- For samples incubated at 80℃: Take 10 µl supernatant to a new tube
- Mix with 10 µl SDS sample buffer
- Incubate all samples at 95℃ for 5 min.
- Take 15 µl of each sample and load on SDS-gel
- Run SDS-gel on 150 V until all samples have wandered into the gel and increase the voltage to 200 V When the loading dye have reached the bottom of the gel, turn it off before it runs out of the gel.
- Wash gel with dH2O and set on a shaking table for 5 min. Repeat 3 times.
- Color with Comassie G250 for an hour. If the coloring isn’t good enough: use Comassie R250 overnight.
- For destaining, a solution of methanol (any grade), 500 ml, dH2O, 400 ml and acetic acid (any grade), 100 ml is needed. Remove the staining solution and replace with the destaining solution, any amount that cover the gel is sufficient. Place on a shaking table for 5-10 mins. Repeat the destaining step until you can read the results.
Colony Screening
Method
- Mix a PCR master mix depending on your polymerase of choice.
- Using a pipette tip to first dip it in a colony.
- Lightly touch the surface of an agar plate with any necessary antibiotics.
- Place the pipette inside an PCR tube with the prepared master mix, and vigorously hit the walls to displace bacterial cells in solution.
- Run a normal PCR at desired temperatures for your polymerase and primers.
- Run the PCR products on an agarose gel to evaluate the colony.
HisPur Ni-NTA Spin Columns
Method
For native conditions prepare the following buffers:- Equilibration Buffer: 20 mM sodium phosphate,300 mM sodium chloride (PBS) with 10 mM imidazole; pH 7.4
- Wash Buffer: PBS with 25 mM imidazole; pH 7.4
- Elution Buffer: PBS with 250 mM imidazole; pH 7.4
Method
Note: The total volume of the 0.2, 1 and 3 mL columns are 1, 8 and 22 mL, respectively. If a sample volume is greater than the column, perform multiple applications and centrifugations until the entire sample has been processed. Be careful not to exceed the resin’s binding capacity. The HisPur Ni-NTA Spin Columns also may be used for gravity-flow purifications.- Equilibrate column(s) to working temperature. Perform purifications at room temperature or at 4 °C.
- Prepare sample by mixing the protein extract with an equal volume of Equilibration Buffer. Use the Equilibration Buffer to adjust the total volume to be ≥ 2 resin-bed volumes.
- Remove the bottom tab from the HisPur Ni-NTA Spin Column by gently twisting. Place column into a centrifuge tube. Note:
- Use 2.0, 15 or 50 mL centrifuge tubes for the 0.2, 1 and 3 mL spin columns, respectively.
- Centrifuge column at 700 × g for 2 minutes to remove storage buffer.
- Equilibrate column with two resin-bed volumes of Equilibration Buffer. Allow buffer to enter the resin bed.
- Centrifuge column at 700 × g for 2 minutes to remove buffer.
- Place the bottom plug in the column and add the prepared protein extract. Mix on an orbital shaker or end-over-end mixer for 30 minutes at room temperature or 4°C.
- Remove the bottom plug. Centrifuge the column at 700 × g for 2 minutes and collect the flow-through in a centrifuge tube.
- Wash resin with two resin-bed volumes of Wash Buffer. Centrifuge at 700 × g for 2 minutes and collect fraction in a centrifuge tube. Repeat this step two more times collecting each fraction in a separate centrifuge tube.
- Elute His-tagged proteins from the resin by adding one resin-bed volume of Elution Buffer. Centrifuge at 700 × g for 2 minutes. Repeat this step two more times, collecting each fraction in a separate tube.
- Monitor protein elution by measuring the absorbance of the fractions at 280nm or by Coomassie Plus (Bradford) Assay Reagent (Product No. 23238). The eluted protein can be directly analyzed by SDS-PAGE.