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Wet Lab: Protocols: Purification of GFPmut3b

Aims: To construct our calibration curve for [GFPmut3b] vs fluorescence we need to obtain a sample of GFPmut3b. This type of GFP is not commercially available and so we will have to construct an expression vector, express it and then purify. To clone the GFP into a vector we used a Biobrick containing the mut3bGFP gene and cloned it into a new vector, with the addition of a His tag. This vector was then transformed into BL21 strains and induced to express the vector.

Reagents

Ni-Buffer A (500ml)

• 50mM Tris pH 7.5 (16.6ml)
• 100mM NaCl (10ml)
• 5% glycerol (25ml)
• 0.1nM PMSF (200ul)
• 2mM B-mercaptoethanol

Ni-Buffer B (500ml)

• 0.5M Imidozole pH 8.0 (17g)
• 100nM NaCl (10ml)
• 5% glycerol (25ml)
• 0.1nM PMSF (200ul)
• 2mM B-mercaptoethanol

Equipment

• Sonicator

Reagents

• Lysozyme solution
• Dnase I

Protocol

1. Thaw the cells slowly on ice and once thawed re-suspend in 10ml of buffer A. To do this use a stripette (10ml) and repeatedly suck up and down.
2. Once fully re-suspended sonicate the cells with the medium probe for 2 x 4mins at 40% power with a 2 second pulse. When sonicating remember to place the tube containing the suspension on ice to prevent any heat transfer denaturing our protein of interest. When the cells are lysed the suspension will appear clearer.
3. Add 0.1ml of 50mg/ml of freshly made Lysozyme solution to the cell lysate together with 0.14ml 1M MgCl2 and 40ul of 10mg/ml DnaseI.
4. Leave at constant temperature for 20 minutes until the solution becomes non viscous.
5. Spin the suspension at 18,000rpm at 4oC for 45minutes.
6. Prepare the column by washing with Ni-Buffer A and load into the column.
7. INSERT COLUMN SETTINGS.
8. Once the the column has been run check the samples for a green color. The sample containing the GFPmut3b should be obvious. Otherwise, check the elution profile and run an SDS-PAGE with any of the samples containing a peak.
9. Once the sample containing the GFPmut3b has been identified we need to measure the concentration. To do this follow the standard protocol for the Bradford Assay.