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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 10^8 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 20mL cold 100mM CaCl2 (10% glycerol), and incubate on ice for 3hr.
5. Harvest cells by cetrifugation. Discard supernatant.
6. Gently resuspend cells in 0.5mL cold 100mL 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 50 seconds.
   • Add appropriate amount of nuclease free water to make a 100μM solution, from which 50μL of 20μM primer solution was 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 dd water
• 5μL buffer
• 0.5μL dNTP (10μM)
• 1μL left primer
• 1μL right primer
• 0.6μL DMSO
• 0.5μL DNA polymerase
3. Run PCR tubes on following thermal cycle program
rsaA

Step	Temperature (°C)	Time (s)
1	98	60
2	98	10
3	60	30
4	72	30
repeat step 2 to 4 for 5 times
5	98	10
6	72.1	30
7	72	30
repeat step 5 to 7 for 25 times
8	72	300
9	4	hold


esp

Step	Temperature (°C)	Time (s)
1	98	60
2	98	10
3	41.2	30
4	72	30
repeat step 2 to 4 for 5 times
5	98	10
6	68.4	30
7	72	30
repeat step 5 to 7 for 25 times
8	72	300
9	4	hold

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

DNA Purification

1. Retrieved from "http://2011.igem.org/Team:Grinnell/Notebook/Protocols"