Team:Washington/Notebook

A DETAILED DESCRIPTION OF THE PROTCOLS WE USED

• Gene Synthesis
• Colony PCR
• Assembly
• Cloning
• Expression
• Purification

Protein Gel

1. Set up overnights of parts 48-51
2. Dilute 1 ul overnight into 1ml
3. Add 1 mm IPTG and let grow for four hours
4. After cells have all grown up uniformly start boiling water for the boil step
5. Add 100uL of overnight to a 1.5mL tube
6. Pellet by spinning at max speed for 30 secs in the microcentrifuge
7. discard supernatent
8. Pull an aliquot of 5x sample loading buffer out of the freezer and thaw
9. Add 20uL BME to aliquot
10. Resuspend samples in 50uL sample loading buffer (pipette up/down)
11. Boil samples for 10 minutes
12. While boiling, prepare 500mL 1x SDS buffer:
    1. 50mL 10x buffer to 450mL water
    2. Take a gel out of the fridge and and put it in the gel box (keep the gel container for staining!!!)
    3. Pour the mixed buffer solution into the half of the gel box that the gel is in
    4. Remove the gel comb
    5. Fill the little container on the top of the gel until it's about 0.5 cm from the top with buffer
    6. Remove any bubbles in the wells
13. Spin down samples for a few seconds
14. Vortex samples
15. Load 3uL into each well
16. Load 10uL of ladder in appropriate wells
17. Run at 180V until the dye is about to fall off the gel

Microscope

1. Set up overnights of parts 48-51. Let grow overnight.
2. Dilute 1 ul overnight into 1ml
3. Add 1 mm IPTG and let grow for four hours
4. After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
5. Also place 1 ul beads in 1 ml along with 1 ul flourophore.
6. Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
7. Next place the cells and the beads under the microscope

Flow Cytometry

1. Set up overnights of parts 48-51. Let grow overnight.
2. Dilute 1 ul overnight into 1ml
3. Add 1 mm IPTG and let grow for four hours
4. After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
5. Also place 1 ul beads in 1 ml along with 1 ul flourophore.
6. Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
7. Read the samples through the flow cytometer

Supernatant Protein Purification, 50mL =

1. Inoculate 50mL culture of TB with ~750uL overnight culture
2. Grow a 37c until OD600: 0.4
3. Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
4. Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
5. Transfer culture to 50mL Falcon Tube
6. Centrifuge at 8000rpm for 20 min to pellet cells
7. Pour supernatant into 60mL syringe with 0.45uM filter attached
8. Apply supernatant to Ni-column (see Ni-column Set up for directions)

Ni-column Set up

1. Transfer 2mL Ni-agarose beads (Quigen) to column
2. wash column with 10mL dH2O
3. Equillibrate column with 10mL running buffer (usually PBS)
4. To reuse column
   1. Wash with 12mL dH2O
   2. Wash with 12mL 100mM EDTA
   3. Wash with 12mL dH2O
   4. wash with 12mL 100mM Ni(SO4)
   5. Wash with 12mL dH2O
   6. Add 12mL 20% Ethanol run till ~5mL remains in column
   7. cap for use later

A ROUGH TIMELINE OF THE PROJECT!