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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 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 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

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.

DNA Purification by Centrifugation

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 rt for 1min, then centrifuge as in step 3.
8. Discard the minicolumn and chill the microcentrifuge tube that contains the eluted DNA.