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From 2011.igem.org

Competent Cells

1. Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10⁸ cells/mL, OD₆₀₀ = 0.3-0.5).
2. Chill cells on ice for 15-120min.
3. Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4°C. Discard supernatant.
4. Completely resuspend cells in cold 100mM CaCl₂ (10% glycerol) up to two thirds full of the bottle, and incubate on ice for 3hr.
5. Harvest cells by cetrifugation. Discard supernatant.
6. Gently resuspend cells in 5mL cold 100mM CaCl₂ (10% glycerol). Incubate on ice for at least 1hr and freeze at -80°C.

Plasmid Transformation

1. Thaw 100μL aliquots of competent cells on ice.
2. Add 10μL DNA to cells.
3. Incubate tubes on ice for 30min.
4. Incubate tubes at 42° C for 90sec.
5. Incubate tubes on ice for 2min.
6. Add 300μL LB to cells and incubate shaking at 37° C for 1hr.
7. Spread cells on selective media
8. Incubate plates overnight at 37° C.

Isolation of DNA for Colony PCR

GeneReleaser is a proprietary reagent that releases DNA from cells while sequestering cell lysis products that might inhibit DNA polymerases.

1. Resuspend the GeneReleaser through inversion, not vortexing. Add 20μL GeneReleaser to each PCR tube.
2. Add cells from plates with a sterile pipette tip with 10μL of appropriate liquid media OR 10μL from overnight liquid culture.
3. Run PCR tubes on following thermal cycle program:

Temperature (°C)	Time (sec)
65	30
8	30
65	90
97	180
8	60
65	180
97	60
65	60
80	hold

4. DNA will be in the clear liquid above the white precipitate at bottom of tube.

Agarose Gel Electrophoresis

1. To make a 0.7% agarose content gel first add 0.21g agarose and then 30mL 1 X TBE buffer to a 250mL Erlenmeyer flask.
2. Microwave until the solution boils, about 45-60sec. Let boil for 5sec, then check for agarose that has not gone into solution. If there is undissolved agarose, boil for 5sec at a time until solution is homogeneous.
3. Let solution sit until it is cool enough to touch and then add 2μL ethidium bromide using caution and swirl mixture.
4. Set up gel tray and combs and pour gel until it is solidified, about 30min.
5. Place gel in chamber oriented with positive electrode at the bottom of the gel and cover with 1X TBE.
6. Add 5μL water, 5μL DNA, and 2μL 6X loading dye.
7. Remove the comb and load each sample along with 10μL of a 1kb ladder. Run at 100 volts.
8. When loading dye has run to the end of the gel, remove gel.

Colony PCR

1. Prepare primers as followed
   • Spin down at 13300 rpm for 50sec.
   • Add appropriate amount of nuclease free water to make a 100μM stock solution, from which a 20μM working solution is made.
2. Make the solution for PCR according to the following recipe
• The resulting mixture we got from DNA isolation (DNA in the clear liquid above and GeneReleaser at the bottom)
• 6.5μL nuclease free water
• 5μL Phusion HF or GC Buffer
• 0.5μL dNTP (10μM)
• 1μL left primer
• 1μL right primer
• 0.6μL DMSO
• 0.5μL Phusion DNA polymerase
3. Run PCR tubes on following thermal cycle program

Step	Temperature (°C)	Time (sec)
1	98	60
2	98	10
3	3°C above the Tm of the primer (without prefix/suffix) that has the lower Tm of the two	30
4	72	extention rate at 30 sec/kb
	repeat step 2 to 4 for 5 times
5	98	10
6	3°C above the Tm of the primer (with prefix/suffix) that has the lower Tm of the two	30
7	72	extention rate at 30 sec/kb
	repeat step 5 to 7 for 25 times
8	72	300
9	4	hold




DNA sample	Temperature used in step 3(°C)	Temperature used in step 6(°C)	Time used for extention steps(°C)
rsaA	60	72.1	30
esp	41.2	68.4	45
rsaA Promotor	61.6	73.4	30
Pxyl	49.2	71.4	30

4. Take amplified DNA from the clear liquid layer on the top.

Purification of DNA > 300bp by Centrifugation

We used the Wizard® SV Gel and PCR Clean-Up System Technical Bulletin from Promega to clean out PCR products >300 nucleotides in length. The protocol is below.

1. Make an SV Minicolumn assembly by placing a minicolumn in a collection tube.
2. Transfer impure DNA solution to minicolumn assembly and incubate at rt for 1min.
3. Centrifuge assembly for 1min at 16,000 x g (14krpm). Remove minicolumn from collection tube and discard liquid in collection tube. Reassemble assembly.
4. Wash minicolumn by adding 700μL Membrane Wash Solution, previously diluted with 95% EtOH, to minicolumn and centrifuging as in step 3. Discard liquid in collection tube.
5. Wash again with 500μL of wash solution, this time centrifuging for 5min at 16,000 x g.
6. Discard liquid in collection tube. Centrifuge for 1min with microcentrifuge lid off or open to allow any remaining EtOH to evaporate.
7. Transfer minicolumn to a clean 1.5mL microcentrifuge tube and add 50μL nuclease-free H₂O to column membrane without touching the membrane with the pipette tip. Incubate at room temperature for 1min, then centrifuge as in step 3.
8. Discard the minicolumn and chill the microcentrifuge tube that contains the eluted DNA.

Gel extraction

We used the same kit from Promega as we used for DNA > 300bp purification. The protocol is slightly different.

1. Find the desired bands on gel under UV and cut off the gel containing these bands.
2. Weigh the gel slice.
3. Save the gel slice in a 1.5ml microcentrifuge tube.
4. Add 1μL Membrance Binding Solution per mg of gel slice.
5. Vortex and incubate at 55°C until gel slice is completely dissolved.
6. Treat the gel mixture as the same as PCR product, follow the DNA > 300bp purification protocol to finish the rest of the gel extraction.

Purification of DNA < 300bp by Centrifugation

1. Estimate the volume of DNA solution.
2. Adjust the concentration of monovalent cations by addition of sodium acetate (0.3M).
3. Mix well. Add 2 volumes ice cold ethanol and mix well. Store in -20°C freezer for 30 minutes.
4. Centrifuge at 0°C taking care of the orientation of the tubes because the DNA pellet will be invisible.
5. Remove supernatant.
6. Fill tube halfway with 70% ethanol and centrifuge at maximum speed for 2 minutes at 4°C.
7. Remove supernatant.
8. Store tube open in a heat block to evaporate the fluid off.
9. Dissolve DNA pellet with buffer from Miniprep kit.

Miniprep to Obtain Plasmid DNA from Overnight Culture

We used the PureYield™ Plasmid Miniprep System from Promega to obtain Plasmid from overnight cultures. The protocol is below.

1. Add 600μL of bacterial culture to a 1.5mL microcentrifuge tube.
2. Add 100μL of Cell Lysis Buffer, and mix by inverting tube 6 times.
3. Add 350μL of cold Neutralization Solution, and mix by inversion.
4. Centrifuge at maximum speed for 3 minutes.
5. Transfer supernatant to PureYield Minicolumn and Collection Tube and centrifuge at maximum speed for 15 seconds. Discard the flow-through.
6. Add 200μL Endotoxin Removal Wash and centrifuge at maximum speed for 15 seconds.
7. Add 400μL Column Wash Solution and centrifuge at maximum speed for 30 seconds.
8. Add 30μL nuclease free water to column and let stand for 1 minute before centrifuging into a 1.5mL cnetrifuge tube.

Freeze-Thaw Cell Lyse

As a preparation of template DNA for colony PCR

1. Add 10μL nuclease-free water to a PCR tube.
2. Inoculate tubes with some cells (generally from plate cultures).
3. Freeze cells at -20°C or -80°C for 10 to 20 minutes.
4. Transfer cells to hot block or thermocycler set at 95°C for 10 minutes.

This method has a lower success rate than using GeneReleaser, but is well suited to doing larger numbers of samples simultaneously.

Conjugation: Transfer desired plamid from E. coli to Caulobacter

After obtaining pMR10 plasmid (can express in both E. coli and Caulobacter) that contains promotor and desired protein gene in E. coli, we will need to transfer the plasmid from E. coli to Caulobacter through conjugation.

1. Prepare liquid overnight cultures of recipient (e.g. Caulobacter), donor (e.g. E. coli with pMR10 plasmid) and helper strains (e.g. E. coli KR2515).
2. Add 600μL of recipient culture and 80μL of both helper and donor strains in a 1.5mL microcentrifuge tube.
3. Spin at 7000 RPM for 1 min and then remove the supernatant.
4. Gently suspend the cells in 1mL PYE (no vortexing).
5. Spin again as above, remove supernatant and resuspend in 25μL PYE.
6. Pipette all concentrate cell culture on a plain PYE plate (w/o spreading) and incubate at 30°C for 5h to overnight.
7. Streak some of the big colony growth from the plain PYE plate out on a PYE plate containing nalidixic acid and kanamycin.
8. Nalidixic acid will kill remaining E. coli but leave the Caulobacter, and kanamycin will select for those cells that have pMR10 plasmid in them.