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From 2011.igem.org

Protocols

Competent Cells

1. Inoculate 500mL LB with 2mL overnight culture. Incubate with shaking to early log phase (~5 x 10⁸ cells/mL, OD₆₀₀ = 0.2-0.4).
2. Chill cells on ice for 15-120min (generally ~30min).
3. Pellet cells in a prechilled sterile centrifuge tube by centrifugation at 5-8krpm for 5min at 4°C. Discard supernatant.
4. Fill centrifuge tube about two thirds full with cold 100mM CaCl₂ (+10% glycerol) and gently but completely resuspend cells; incubate on ice for 3hr or overnight.
5. Harvest cells by cetrifugation as before. Discard supernatant.
6. Gently resuspend cells in 5mL cold 100mM CaCl₂ (+10% glycerol). Incubate on ice for at least 1hr. Aliquot into 100μL aliquots, then flash freeze and store at -80°C.

Plasmid Transformation by Heat Shock

1. Thaw 100μL aliquots of competent cells on ice.
2. Add 10μL DNA to cells.
3. Incubate tubes on ice for 30min.
4. Heat shock 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 1-2hrs.
7. Spread 200μL cells on selective media.
8. Incubate plates overnight at 37° C.

Isolation of DNA for Colony PCR using GeneReleaser

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 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 5μL of selected ladder. Run at ~100V (lower voltage for clearer bands but slower run time).
8. When loading dye has run about two thirds of the gel, remove gel and image with UV.

Colony PCR

1. Prepare primers as follows:
   1. Spin down at 13300 rpm for 50sec.
   2. 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 (either add directly to GeneReleaser product w/o disturbing the precipitate OR use 8μL of freeze-thaw product).
• 7.65μL nuclease free water (9.65μL if using freeze-thaw product)
• 5μL Phusion HF or GC Buffer
• 0.5μL dNTP (10μM)
• 0.5μL left primer (20μM)
• 0.5μL right primer (20μM)
• 0.6μL DMSO
• 0.25μL Phusion DNA polymerase
• For a final volume of 25μL
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 steps 2 through 4 for 5 iterations
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 steps 5 through 7 for 25 iterations
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
PrsaA	61.6	73.4	30
Pxyl	49.2	71.4	30

4. Take amplified DNA from the clear liquid layer on the top (if used GeneReleaser for DNA isolation).


5. Occasionally, we used colony PCR to check if transformation cells have the target gene in them. The recipe for PCR is the same as the aforementioned one but a different thermal cycle program is used.

6. Step	Temperature (°C)	Time (sec)
   1	98	30
   2	98	10
   3	Use 47 for pMR10 plasmid and 55.3 for pSB1C3 plasmid	30
   4	72	extention rate at 30 sec/kb
   	repeat steps 2 through 4 for 30 iterations
   8	72	300
   9	4	hold

Colony PCR with GoTaq® Green Master Mix

We also used GoTaq® Green Master Mix, a product of Promega Corporation.

1. Thaw Master Mix (MM) to rt, vortex, and centrifuge.
2. Prepare rxn mixture on ice:
   • 12.5μL 2X MM
   • 0.25-2.5μL each 10μM primer
   • 1-5μL template DNA (<250ng)
   • Nuclease-free H₂O to 25μL
3. Add mineral oil (1-2 drops) if your thermocycler does not have a heated lid.
4. Perform PCR (see guidelines below).

This is the generalized PCR cycling protocol for use with GoTaq®

Step	Temp (°C)	Time (sec)
1	95	120
2	95	30 to 60
3	TM - 2	30 to 60
4	72 to 74	60/kb
Repeat steps 2 through 4 25 to 40 times
5	72 to 74	300
6	4	hold

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 >300bp 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. (This step generally also works with the lid on.)
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 rt for 1min, then centrifuge as in step 3.
8. Discard the minicolumn and store the microcentrifuge tube that contains the eluted DNA at -20°C.

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 out the gel containing these bands. (Smaller slices are better. Also, as the UV tends to damage the DNA, minimize the exposure to UV.)
2. Weigh the gel slice.
3. Save the gel slice in a 1.5ml microcentrifuge tube.
4. Add 1μL Membrane 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 the same as PCR product, follow the DNA > 300bp purification protocol to finish the rest of the gel extraction.

Gel extraction 2

Alternatively, we used Zymoclean Gel DNA Recovery Kit from Zymo Research.The protocol is slightly different from the Promega kit.

1. Pre-weigh the 1.5ml microcentrifuge tube.
2. Find the desired bands on gel under UV and cut out the gel containing these bands. (Smaller slices are better. Also, as the UV tends to damage the DNA, minimize the exposure to UV.)
3. Save the gel slice in a 1.5ml microcentrifuge tube.
4. Weigh the microcentrifuge tube and calculate out the mass of the gel slice.
5. Add 300μL ADB per 100mg of gel slice.
6. incubate at 55°C for about 10min until gel slice is completely dissolved.
7. Transfer the melted agarose solution to a column in a collection tube.
8. Add 200μL of Wash Buffer to the column and centrifuge at 16000g for 30 seconds. Discard the flow-through and repeat the wash step.
9. Add >=6μL water directly to the column matrix. Place column into a 1.5ml tube and centrifuge at 16,000g for 30sec to elute DNA.
10. Pure DNA in water now is ready for use.

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 any remaining 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 centrifuge tube.

Alternate Miniprep Protocol

We also used Promega's Wizard® Plus SV Minipreps kit. The original procedure can be found at Promega's website.

1. Pellet overnight culture for 5min. at rt (use 3mL overnight culture if plasmid is low copy, 1.5mL otherwise).
2. Resuspend in 250μL of the provided cell resuspension sol'n.
3. Add 250μL cell lysis sol'n, invert 4 times to mix.
4. Add 10μL alkaline protease sol'n, invert 4 times to mix, let stand at rt 5min.
5. Add 350μL neutralization sol'n, invert 4 times to mix.
6. Centrifuge at top speed 10min at rt.
7. Insert column into collection tube and decant lysate into the column.
8. Centrifuge at top speed for 1min, discard flowthrough and reinsert column into collection tube.
9. Add 750μL column wash sol'n (w/EtOH), centrifuge at top speed 1min, discard flowthrough and reinsert column into collection tube.
10. Repeat previous step with 250μL wash sol'n.
11. Centrifuge at top speed 2min at rt.
12. Transfer to a new 1.5mL microcentrifuge tube, being careful to avoid leaving any wash sol'n on the column.
13. Add 50μL nuclease free H₂O and centrifuge at top speed for 1min at rt.
14. Discard column and store flowthrough at -20°C or below.

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. (Depending on container, this may take 30 to 40min.)
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 replicate 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.

Preparing samples for and running a polyacrylamide gel

1. Inoculate 3mL of broth and let grow overnight.
2. Spin down 1mL of overnight culture.
3. Collect supernatant (~900μL) and perform protein precipitation protocol.
4. Resuspend pellet in 100μL broth.
5. Add 50μL 3X Sample Buffer (aliqiots are 850μL, add 150μL β-mercaptoethanol).
6. Heat tubes of pelleted cells and supernatant with Sample Buffer at 95°C for 3-5 minutes. They can now be frozen at 20°C until further use or used immediately.
7. Load 20μL of sample into each well of a polyacrylamide gel and let gel run for 1 minute.
8. Then load 15-20μL more sample, or however much will fit in the well.
9. Run at 125-200V for 1-2 hours. Make sure the buffer is always covering the wells.
10. When the dye has reached the bottom of the gel, remove from gel rig, open plastic gel container using well comb, and place in bin.
11. Rinse with deionized water once and then let it sit in deionized water for 5 minutes then pour water off then let sit in water again for 5 minutes. Pour off water.
12. Prefix the gel in “fixing” solution (50% MeOH, 10% HoAC, and 40% water) for 30 minutes or overnight shaking slowly.
13. Then stain the gel with Coomassie stain (“fixing” solution with 0.25% Coomassie Blue R-250 added) for 2-4 hours, until the gel is a uniform blue color. Staining is complete when the gel is no longer visible in the dye solution.
14. Destain for 4-24 hours (5% MeOH, 7.5% HoAC, 87.5% water). Bands will begin to appear in 1-2 hours. Destain until background is clear.

Precipitating proteins out of supernatant in preparation for running a protein gel.

1. Add 110μL of 100% TCA to each tube and vortex for 10 seconds.
2. Incubate tubes on ice for 15 minutes.
3. Centrifuge tubes at full speed for 5 minutes in a chilled rotor.
4. Discard supernatant by pouring out of the tube. Pellet will remain at the bottom of the tube. It is important to remove all of the supernatant. Blot the lip of the inverted tube with a Kim Wipe. Spin down the tube again for 5 seconds and use a mciropipettor to remove any remaining supernatant.
5. Resuspend the pellet in 40μL of 0.5 M Tris base or PYE and then add 20μL Sample buffer.