From 2014.igem.org

Brauer Group Protocols

Polymerase Chain Reaction

Phusion HF Polymerase Protocol

Per tube, add in this order:

Cloning water	21.5 µl
5x HF buffer	10 µl
5M Butane	10 µl
DMSO	4 µl
10µM Primers (f+r)	2.5 µl
10µM dNTPs	1.0 µl
Template DNA	0.5 µl
Phusion Polymerase	0.5 µ

PCR Reaction in Thermal Cycler:

98 °C

30 seconds

30 Cycles

98 °C	20 seconds
Annealing Temperature (NEB TM Calculator)	20 seconds
72 °C	Extension Time (20 seconds/kb)

72 °C

5 minutes

4 °C

Forever

Colony PCR

1. Prepare 10 µl of cloning water per colony to be picked in PCR tubes.
2. Prepare culture tube filled with 4mL LB, 4µL resistance
3. Pick colony and resuspend in cloning water by pipetting up and down
4. Next, drop the tip inside the culture tube, or dot onto pre-numbered LB plate
5. Heat water with colonies for 10 minutes in 98°C; use as “template DNA”
6. Put culture tubes in 37°C shaker to incubate (overnight) while PCR is running.
7. Go TAQ PCR mix

Cloning Water	35µl
Vortexed 5x Green Taq Buffer	10µl
10µM Sequencing Primer	2.5µl
10µM dNTPs	1.0µl
Template DNA (heated above)	1.0µl
GoTaq Polymerase	0.5µl

8. TAQ PCR Protocol

95 °C

2 minutes

35 Cycles

95 °C	1 minute
Annealing Temperature (NEB TM Calculator)	45 seconds
72 °C	Extension Time (20 seconds/kb)

72 °C

5 minutes

4 °C

Forever

9. Run Gel Visualization
10. Add 15µl PCR product (no need 6x loading dye)
11. Freeze & Miniprep cultures that have desired bands

Gradient PCR

Annealing Temperature: 5 °C below to 5 °C above.
Example: 62°C = 57°C -> 67°C
Increase Annealing Time to 30 seconds

Making a Gel for Gel Electrophoresis

0.7g of Agarose
Add 100 ml 1x TAE buffer into an Erlenmeyer flask
Microwave for 2 minutes
Cool down under tap water for 20 seconds
Add 5 µl Sybrsafe
Pour the gel into mold with comb. Let cool for 30 minutes.
Remove the comb.
Transfer tray into an electrophoresis chamber, wells facing black side (anode) of chamber
Fill chamber with 1x TAE until TAE just covers the gel

Gel visualization/extraction

1. Prepare samples

Visualization	Extraction
10µl PCR Product	50µl PCR Product
2µl 6x Loading Dye	10µl 6x Loading Dye

2. Add DNA ladder Standard into first well.
3. Pipet prepared samples into rest of wells.
4. Seal electrophoresis chamber with chamber cap and plug cables in VWR power unit.
5. Set 125V, Run for 30 minutes (or longer) depending on length of sample expected.
6. Remove Gel tray once finished and visualize using trans-illuminator.
7. Extract if necessary into a preweighed 1.5 ml microcentrifuge tube.

Gel purification Gel purification kit

Using Zymoclean Gel DNA Recovery Kit
1. Add 3 volumes (µl) for each volume of agarose (mg) excised from the gel.
2. Incubate at 55°C for 10 minutes until gel is dissolved
3. Pipette into a Zymo-Spin Column & Collection Tube
4. Centrifuge (at 16000 x g)for 1 minute, and discard the flow-through.
5. Add 200µl DNA wash Buffer and centrifuge for 30 seconds.
6. Add another 200µl DNA wash buffer and centrifuge for a minute.
7. Place the spin column into a new sterile microcentrifuge tube.
8. Add up to 30µl of Cloning Water directly to the column matrix.
9. Let it sit for 4 minutes, and centrifuge for 4 minutes.

Treating with DPNI

1. Add to a PCR rxn mix after PCR.
2. 1µl DPNI per 50 µl rxn mix.
3. Let it run 37°C for 1 hour.

DNA Purification DNA Purification kit

Using Zymoclean DNA Clean & Concentrator
1. Add 5 volumes of DNA binding Buffer to PCR product or Short DNA fragments.
2. Vortex Tube.
3. Load mixture into Zymo-Spin Column and Collection Tube
4. Centrifuge (16000 x g) for 30 seconds. Discard flow-through.
5. Add 200µl of DNA Wash Buffer and centrifuge for 30 seconds.
6. Add another 200µl of DNA Wash Buffer and centrifuge for 2 minutes.
7. Place the spin column into a new sterile microcentrifuge tube.
8. Add up to 30µl of Cloning Water directly to the column matrix.
9. Let it sit for 4 minutes, and centrifuge for 4 minutes.

Golden Gate Digestion/Ligation

1. Design Primers accordingly, taking into account Type IIs Restriction Enzyme site.
2. Add purified pieces you want to ligate based on concentration results (100ng / x ng/µl) into a PCR tube.
3. Add cloning water to the mix up to 10.5µl total.
4. Add 1.5µl Cutsmart Buffer to the mix.
5. Vortex ligase buffer. Add 1.0µl to the mix.
6. Add 1µl of Type IIs restriction enzyme (we usually use sapI)
7. Add 1µl of T4 Ligase to the mix.
8. Total should be 15µl in the tube. If volume of pieces you want to ligate together add up to greater than 10.5µl, scale up the mix proportionally.
Reaction assembly

50 x

37°C	3 min
16°C	4 min

50°C

5 min

80°C

5 min

Restriction Enzyme Digestion

1. Follow NEB protocols.
2. Find appropriate buffer if doing double digests.
3. Heat inactivate at the end.
4. If doing sequential digests, heat inactivate in between each step.

Blunt End Ligation

1. Add 18µl of purified PCR product into a PCR tube.
2. Add 2.25µl of 10x T4 Ligase Buffer
3. Add 1.125µl of T4 PolyNucleotide Kinase.
4. Put in thermocycler for 37°C for 45 minutes.
5. Add 1.125µl T4 DNA Ligase
6. Leave at room temperature for at least 1 hour.
7. Heat inactivate whatever you are not using at 65°C for 20 minutes.

Transformation (Electroporation)

1. Put electroporation cuvette on ice and thaw electro-competent cells for 5 minutes.
2. Prepare 500µl LB and a labeled culture tube.
3. Add up to 5µl of DNA directly to the competent cells. (usually use 2µl)
4. Transfer mix into cuvette, ensuring no bubbles and mix touching both sides.
5. Wipe down cuvette to remove moisture.
6. Place in electroporation machine and start.
7. If arc occurs, try with smaller amounts of DNA.
8. If successful, immediately add the LB directly into the cuvette.
9. Transfer LB + transformed cells into the culture tube.
10. Record time and incubate in the 37°C shaker for 1 hour.

Plating

1. Prepare two appropriate antibiotic resistance “100µl” and “Rest” Plating

2. Add 100µl of transformed culture (after the 1 hour incubation) onto the 100µl plate.
3. Use a sterile spreader to streak the liquid culture onto the plate in the order on the left:
4. On the “Rest” Plate, pour the rest of the liquid culture and spread evenly on the plate.
5. Flip and put in the 37°C incubator overnight (no longer than 20 hours).
6. Wrap your plate after sufficiently grown and store in the 4°C fridge for up to 45 days.

Liquid Cultures

1. Determine volume of culture to grow in a culture tube. We usually use 4ml of LB.
2. In the biosafety hood, add 4ml of LB, and 4µl of each appropriate resistance(s).
3. Use a tip to pick up a single colony from the plate.
4. Drop the tip into the media.
5. Cap the culture tube to the first stop.
6. Incubate in 37°C shaker at 250rpm for up to 16 hours.

Freeze Cultures

1. Add 500µl of 30% glycerol into a cryo-freeze tube.
2. Add 500µl of overnight culture.
3. Store in -80°C.

Plasmid Miniprep

Using Zyppy Plasmid Miniprep Kit

1.Centrifuge overnight culture for 10 minutes at 5000 x g
2. Discard supernatant. Resuspend with 550µl LB
3. Transfer culture to 1.5 ml microcentrifuge tube.
4. Add 100 µl of 7X Lysis Buffer (Blue). Shake well.
5. Within 2 minutes, add 350 µl of cold Neutralization Buffer (Yellow). Shake well.
6. Centrifuge at 16000 x g for 4 minutes.
7. Transfer supernatant into a Zymo-Spin Column and Collection Tube.
8. Centrifuge at 16000 x g for 30 seconds. Discard flow-through.
9. Add 200µl Endo-Wash Buffer. Centrifuge at 16000 x g for 30 seconds.
10. Add 400µl Zyppy Wash Buffer. Centrifuge at 16000 x g for 2 minutes.
11. Transfer column into new labeled microcentrifuge tube.
12. Add 30µl of cloning water directly to column matrix. Let sit for 4 minutes.
13. Centrifuge at 16000 x g for 4 minutes to elute.

Light Induction Experiment

1. Co-transform chromophore plasmid with light sensor (2µl of each miniprepped product). Light Induction Expt

2. Plate and pick colonies.
3. Start liquid cultures with appropriate antibiotic resistance for light sensor(s) and appropriate controls.
4. Wrap tubes that you want to use in dark with foil carefully.
5. Freeze experimental stock for future use.
6. Measure OD600
7. Dilute to OD 0.1
8. Grow for 2 hours in 37°C shaker.
9. Transfer into deep well plates and induce with No and Max ATC in both Light and Dark conditions (2 controls, light and ATC).
10. Grow for 6-8 hours in a lighted growth chamber (wrap plates that will be in the dark).
11. Spin down colonies at 3000x g for 15 minutes.
12. Resuspend with 200µl 1 x PBS.
13. Measure Fluorescence/Absorbtion in 96 well plates.
14. For EYFP we used Excitation and Emission values of 485nm and 528nm respectively.

Rebstock Group Protocols

Electroporation Protocol

Take 1 mL from tube culture and transfer it into 50 mL of LB broth in a flask. Let culture for 2 hrs in the 37°C room.
Pipet 150 μL of each flask into a well in a 96 well plate and analyze the optical density. May proceed when OD600 is between 0.1 and 0.15
Chill the flasks for 15 min. and shake/swirl every 5 min.
Empty the flasks into centrifuge tubes
Centrifuge for 5 mins at 4000 rpm.
Extract supernatant with vacuum and fill with autoclaved water to between 15-20 mL--just make sure they are all even.
Repeat step 5
Repeat step 6
Repeat step 5
Add 200 μL of 10% glycerol into each tube
Pipet 50 μL of each into a cuvette
Pipet 4 μL of plasmid suspension into the cuvette and ice for 10 min.
Transfer to electroporation cuvette--be sure not to touch the metal--and keep on ice
Perform electroporation under the second E. coli setting: 2500 V with a 0.2 cm cuvette
Transfer 1 mL of LB to each electroporation cuvette.
Transfer all of the contents of the cuvettes into tubes. Grow for 1 hr at 37°C
Transfer the contents of the tubes into centrifugable cuvettes
Plate varying amounts of the cell suspension. **For large volumes (>500 μL) spin down the desired volume in a small centrifuge, then remove most of the supernatant so that the volume is ~150 μL. Resuspend the cells and plate the remaining suspension.
Make sure the plates are dry before storing them at 37°C overnight or until discernible colonies form.

Inoculation of Transformed E. coli from Plates into Tubes Protocol innoculating media

Prepare all labels before hand--be sure to include culture identifiers, the date, and your initials
Light a bunsen burner and perform all steps using sterile technique
Pipet 4 mL of LB into each tube using a volumetric pipet
Using a 10 μL pipettman, add 4 μL of the appropriate antibiotic to each tube
Now put a tip on your 10 μL pipettman and scrape a single colony from your plate
Eject the tip into the tube and cap the tube
Place all tubes on the shaker table in the 37°C room

Recipe for 10X M9 Stock Solution for Nitrogenase Activity Assay (100mL):

Reagents:

0.026 g CaCl2·H2O,
0.030 g MgSO4,
10.4 g Na2HPO4,
3.4 g KH2PO4,
4 g glucose

Procedure:

Fill a clean 100 mL beaker with ~70 mL of water
Add a magnetic stir bar and place on stir plate, stirring moderately
Add the appropriate amount of reagent listed above IN THE ORDER listed above
Increase or decrease the speed of the stir plate until all particles are dissolved
Once the solution is clear and colorless, remove the stir bar and pour the contents of the beaker into a 100 mL graduated cylinder
Fill with water to the 100 mL mark
Pour the solution back into the original beaker
In a hood with a flame, filter the solution with a sterile filter into an autoclaved bottle
Label and store for future use

Recipe for 1000X M9 Supplemental Stock Solution (100 mL) Storing solutions

Reagents:

0.3 g MnSO4,
7.6 g Na2MoO4*2H2O,
0.010 g p-aminobenzoic acid,
0.005 g biotin

Procedure:

Fill a 100 mL beaker with ~70 mL of water
Add a magnetic stir bar and place on stir plate, stirring moderately
Add the appropriate amount of reagent listed above
Increase or decrease the speed of the stir plate until all particles are dissolved
Remove the stir bar and pour the contents of the beaker into a 100 mL graduated cylinder
Fill with water to the 100 mL mark
Pour the solution back into the original beaker
In a hood with a flame, filter the soution with a sterile filter into an autoclaved bottle
Label and store for future use

Recipe for 100X Ferric Citrate Stock Solution (100 mL)

Reagent:

0.36 g Ferric citrate

Procedure:

Fill a 100 mL beaker with ~70 mL of water
Add a magnetic stir bar and place on a stir plate, stirring moderately
Add the appropriate amount of reagent listed above
Increase or decrease the speed of the stir plate until the surface of the particles have dissolved--not all of the rusty-colored particles will dissolve
Transfer the suspension into an autoclaveable bottle
Autoclave
Once removed from the autoclave, the particles should be dissolved and the solution should be a yellow-brown color.
Label and store for future use

Recipe for Other 1X K. pneumoniae Medium (100 mL)

Mix the following to make 100 mL of solution:

0.01g CaCl22H2O
0.025g MgSO47H2O
2.5 g Na2HPO4
0.3g KH2PO4
0.1g NaCl
0.00029 g FeCl3
0.000025 g Na2MoO42H2O
2.0 g sucrose
0.6 mL (per liter) of 22% (wt/vol) NH4Ac

Preparation for Acetylene Reduction Assay GC machine

Grow engineered E. coli strains at 37°C overnight in 4 mL of LB broth with antibiotics
Collect cultures by centrifugation and wash with sterilized water three times
Resuspend cells in 5 mL of nitrogen-deficient medium with 10 mM glutamate as a nitrogen source in a sealed 25 mL tube and grow to a final OD600 of 0.2-0.4.
Then evacuate the headspace and flush with argon.

Preparation for Gel Electrophoresis

Select an appropriately sized plate for the number of samples you would like to run
Place the two ends into the plate to prevent the liquid gel from running
Prepare the gel as follows: for a 1% gel, pour 100 mL of 1X TAE and 1g of agarose into a 250 mL flask
Microwave until it begins to bubble.
Remove with a glove or hot hand and swirl until it becomes sort of cloudy again
Repeat steps 4 and 5 until the liquid is clear upon removal from microwave
Then add 1.5 μL of ethidium bromide for every 50 mL of gel solution--may want to allow the flask to cool to the touch before adding. Swirl to mix.
Set the comb for the appropriate number of lanes into the plate--be sure that the comb does not touch the bottom of the plate
Pour the gel
Allow to set for about 30 minutes.

CPEC (Circular Polymerase Extension Cloning) Protocol – Andrew Ng

Design primers that include overhangs for fragments that you want to assemble by hand or using a program such as Snapgene (these primers can also be used for Gibson assembly)
Using Phusion polymerase, PCR each insert as well as your plasmid backbone and verify on a gel.

IMPORTANT- it is highly recommended that you optimize your PCR protocol (either using temperature gradient or DMSO) so that only one band can be seen in your PCR

Digest PCR product using DpnI (digests methylated DNA)

If PCR was performed using Phusion polymerase from NEB, you can

Add the DpnI enzyme from NEB directly. For this, I add 1 ul of enzyme to 50 ul of PCR product and incubate for 3 hours or more 4. PCR purify your inserts,

NOTE: if you were unable to get a single band in your PCR, you might consider gel purification if you can get enough yield

Using a 2:1 molar ratio of insert to vector (if you did not PCR purify, I would just eyeball concentrations based on length of parts and darkness of band in gel), make your CPEC reaction as follows:

4 ul HF Buffer
0.4 ul dNTPs
.1 ul Phusion Polymerase
Insert
Vector
Fill to 20 ul with dH20

(Note for this step: The more DNA you have available to use here the better. I would recommend using a maximum of between 8-10 ul of DNA in total though due to the salt content)
Use the following thermal cycler protocol (or just navigate to CPEC in the folder labeled Andrew in the BioRad C1000)

98.0C for 30s
98.0C for 10s
Ramp to 55.0C at a rate of 0.1C/s
55.0C for 30s
72.0 C for 30s/kb of construct
Repeat 1-5 for 10-30 cycles (depending on the “complexity” of your assembly)
g. 72.0 C for 10 min

Transform cells with 2-5ul of CPEC mix

Team:WashU StLouis/Protocol

From 2014.igem.org

Protocols

Brauer Group Protocols

Rebstock Group Protocols

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