Team:Washington/Notebook/IMAC protocol

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Revision as of 00:30, 17 October 2009

BioRad Micro Column Protein Prep Protocol

Day 1: Overnights
- Pick a single colony from plate and inoculate 1mL fresh media, with proper antibiotic, in 15mL culture tube
- Place lid on tube as not to form an air tight seal
- Shake at 37C for 16-24 hours at 200+ rpm
Day 2: Expression
- Inoculate 50mL media with 500uL over night, in 250mL flask
- Shake 250mL flask at 37C at 200+ rpm
- Monitor OD600 of culture until OD600 ~0.4
  - Use 200uL of culture and 800uL water in 1mL cuvette
  - Read OD600 and multiply by 5 t get original OD600
  - Should take ~2-3 hours
- INDUCE culture once culture gets to OD600 ~0.4 with IPTG so that the final concentration of IPTG is 0.5mM (ie 25uL 1M IPG for 50mL culture)
- Place in shaker 24-36hr at 18C.
Day 3: STORE
- Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
- Store cells in -20C until ready for purification
Day 3/4: Purification
- Lysis (~1hr)
  - Add 1mL of lysis buffer, re-suspend, transfer to a 2mL eppindorf tube
  - Continue to incubate at room temperature for 20min, mixing with plate mixer
    - If worried about protein stability then incubate in cold room for 1hr on rocker
  - Spin the lysis for 30-60min at 15000rpm (spin longer if supernatant is not clear, but 1hr should be plenty!)
  - Transfer supernatant to a fresh tube (~1800uL)
- Purification (~1hr)
  - Add 100uL of NiNTA Agarose 50% slurry to each column (this is the Ni-beads)
  - Add 600uL of diH2O to each column
  - Spin 500rpm for 10s, discard flow through
  - Add 500uL of wash buffer, spin, discard flow through
  - Add 500uL of supernatant, spin, discard flow through
    - Repeat previous step until all supernatant has passed over column (~4x)
  - CHANGE TO FRESH COLLECTION TUBE
  - Add 200uL of elution buffer pipette up and down to mix the beads
  - Incubate for 2min the spin at 500rpm for 1min
  - THE FLOW THROUGH IS YOU PURIFIED PROTEIN!

@@ Line 1: / Line 1: @@
 ====BioRad Micro Column Protein Prep Protocol====
+*Day 1: Overnights
+**Pick a single colony from plate and inoculate 1mL fresh media, with proper antibiotic, in 15mL culture tube
+**Place lid on tube as not to form an air tight seal
+**Shake at 37C for 16-24 hours at 200+ rpm
+*Day 2: Expression
+**Inoculate 50mL media with 500uL over night, in 250mL flask
+**Shake 250mL flask at 37C at 200+ rpm
+**Monitor OD600 of culture until OD600 ~0.4
+***Use 200uL of culture and 800uL water in 1mL cuvette
+***Read OD600 and multiply by 5 t get original OD600
+***Should take ~2-3 hours
+**INDUCE culture once culture gets to OD600 ~0.4 with IPTG so that the final concentration of IPTG is 0.5mM (ie 25uL 1M IPG for 50mL culture)
+**Place in shaker 24-36hr at 18C.
+*Day 3: STORE
+**Spin down cells at 4000rpm for 20 min in 50mL falcon tubes, discard supernatant
+**Store cells in -20C until ready for purification
+*Day 3/4: Purification
+**Lysis (~1hr)
+***Add 1mL of lysis buffer, re-suspend, transfer to a 2mL eppindorf tube
+***Continue to incubate at room temperature for 20min, mixing with plate mixer
+****If worried about protein stability then incubate in cold room for 1hr on rocker
+***Spin the lysis for 30-60min at 15000rpm (spin longer if supernatant is not clear, but 1hr should be plenty!)
+***Transfer supernatant to a fresh tube (~1800uL)
+**Purification (~1hr)
+***Add 100uL of NiNTA Agarose 50% slurry to each column (this is the Ni-beads)
+***Add 600uL of diH2O to each column
+***Spin '''500rpm for 10s,''' discard flow through
+***Add 500uL of '''wash buffer,''' spin, discard flow through
+***Add 500uL of '''supernatant,''' spin, discard flow through
+****Repeat previous step until all supernatant has passed over column (~4x)
+***CHANGE TO FRESH COLLECTION TUBE
+***Add 200uL of elution buffer pipette up and down to mix the beads
+***Incubate for 2min the spin at 500rpm for 1min
+***'''THE FLOW THROUGH IS YOU PURIFIED PROTEIN!'''