Team:Washington/Notebook
From 2009.igem.org
(Difference between revisions)
Line 99: | Line 99: | ||
##Add 500uL supernatant to the column | ##Add 500uL supernatant to the column | ||
##Spin for 30sec at 500rpm | ##Spin for 30sec at 500rpm | ||
- | + | ##Discard flow through | |
+ | ##Repeat 1-3 until all supernatant has run though the column | ||
+ | #Wash column | ||
+ | ##Apply 500uL PBS to column, aspirate to suspend beads | ||
+ | ##Spin column for 30sec at 500rpm, discard flow through | ||
+ | ##Repeat 1-2 | ||
+ | #Elute protein off of column | ||
+ | ##Make PBS with 1mg/mL BSA and 100uM imidazole | ||
+ | ##Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads | ||
+ | ##Let sit for 2 min | ||
+ | #Place Column in clean collection tube | ||
+ | #Spin column for 5min at 500rpm | ||
+ | #FLOW THROUGH IS YOUR PURIFIED PROTEIN | ||
Revision as of 19:44, 13 October 2009
A DETAILED DESCRIPTION OF THE PROTCOLS WE USED
- Gene Synthesis
- Colony PCR
- Assembly
- Cloning
- Expression
- Purification
Protein Gel
- Set up overnights of parts 48-51
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have all grown up uniformly start boiling water for the boil step
- Add 100uL of overnight to a 1.5mL tube
- Pellet by spinning at max speed for 30 secs in the microcentrifuge
- discard supernatent
- Pull an aliquot of 5x sample loading buffer out of the freezer and thaw
- Add 20uL BME to aliquot
- Resuspend samples in 50uL sample loading buffer (pipette up/down)
- Boil samples for 10 minutes
- While boiling, prepare 500mL 1x SDS buffer:
- 50mL 10x buffer to 450mL water
- Take a gel out of the fridge and and put it in the gel box (keep the gel container for staining!!!)
- Pour the mixed buffer solution into the half of the gel box that the gel is in
- Remove the gel comb
- Fill the little container on the top of the gel until it's about 0.5 cm from the top with buffer
- Remove any bubbles in the wells
- Spin down samples for a few seconds
- Vortex samples
- Load 3uL into each well
- Load 10uL of ladder in appropriate wells
- Run at 180V until the dye is about to fall off the gel
Microscope
- Set up overnights of parts 48-51. Let grow overnight.
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
- Also place 1 ul beads in 1 ml along with 1 ul flourophore.
- Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
- Next place the cells and the beads under the microscope
Flow Cytometry
- Set up overnights of parts 48-51. Let grow overnight.
- Dilute 1 ul overnight into 1ml
- Add 1 mm IPTG and let grow for four hours
- After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
- Also place 1 ul beads in 1 ml along with 1 ul flourophore.
- Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
- Read the samples through the flow cytometer
Supernatant Protein Purification, 50mL
- Inoculate 50mL culture of TB with ~750uL overnight culture
- Grow a 37c until OD600: 0.4
- Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
- Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
- Transfer culture to 50mL Falcon Tube
- Centrifuge at 8000rpm for 20 min to pellet cells
- Pour supernatant into 60mL syringe with 0.45uM filter attached
- Apply filtered supernatant to Ni-column (see Ni-column Set up for directions)
- Collect flow through and re-apply to column
- Wash column with 12mL PBS
- Elute column into 15mL condenser tube with 12mL PBS with 100mM imidazole
- Spin elutant in condenser tube until desired volume (~500uL) this is you purified protein.
Ni-column Set up
- Transfer 2mL NiNTA beads (Quigen) to column
- wash column with 10mL dH2O
- Equillibrate column with 10mL running buffer (usually PBS)
- To reuse column
- Wash with 12mL dH2O
- Wash with 12mL 100mM EDTA
- Wash with 12mL dH2O
- wash with 12mL 100mM Ni(SO4)
- Wash with 12mL dH2O
- Add 12mL 20% Ethanol run till ~5mL remains in column
- cap for use later
Supernatant Protein Purification, 2mL
- Inoculate 50mL culture of TB with ~750uL overnight culture
- Grow a 37c until OD600: 0.4
- Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)
- Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant
- Prep NiNTA columns
- Place micro-centrifuge columns in collection tubes
- In micro-centrifuge columns add 200uL NiNTA beads
- Add 500uL PBS, aspirate to thoroughly rinse columns
- Spin columns with collection tubes for 30sec at 500rpm
- Transfer 2mL of growing culture to eppendorf tube
- Spin tube for 20min at 8000 rpm
- Remove supernatant, carefully as to not disrupt the pellet and set aside
- Bind Protein to column
- Add 500uL supernatant to the column
- Spin for 30sec at 500rpm
- Discard flow through
- Repeat 1-3 until all supernatant has run though the column
- Wash column
- Apply 500uL PBS to column, aspirate to suspend beads
- Spin column for 30sec at 500rpm, discard flow through
- Repeat 1-2
- Elute protein off of column
- Make PBS with 1mg/mL BSA and 100uM imidazole
- Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads
- Let sit for 2 min
- Place Column in clean collection tube
- Spin column for 5min at 500rpm
- FLOW THROUGH IS YOUR PURIFIED PROTEIN
A ROUGH TIMELINE OF THE PROJECT!