Team:UCLA/Notebook/Recombinant Expression/21 July 2015
Contents
IMAC Purification of 1L Batch
Samples were heat treated for 10 minutes at 80 degrees in a water bath to resolubilize the proteins, which are soluble at room temperature.
Resin Preparation
1. Add 4 mL of Histidine resin to a 50mL Falcon tube.
2. Add 8 mL of DI water.
3. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Decant supernatant.
4. Add 10 mL (about 2.5 BV) of 1X Binding buffer.
5. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Decant supernatant.
Batch Purification Protocol
1. Dilute 5mL of lysate sample in 5mL of 1X Binding buffer.
2. Add to prepared resin. Spin on rotary spinner for 30 minutes at room temperature.
3. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 1.
4. Add 10mL of Wash buffer (20 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature or until fully incorporated into resin.
5. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 2.
6. Add 10mL of Wash buffer (50 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature.
7. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 3.
8. Add 10mL of Wash buffer (100 mM imidazole) to resin. Spin on rotary spinner for 5 minutes at room temperature.
9. Centrifuge for 2 minutes at 700g at 4 degrees Celsius. Collect supernatant as Fraction 4.
10. Add 10mL of Elution buffer (250 mM imidazole) to resin. Spin on rotary spinner for 10 minutes at room temperature.
11. Run resin contents through a gravity filtration column. Collect eluate as Fraction 5.
12. Add about 6mL (to the top of column) of Strip Buffer. Collect eluate as Strip Fraction.
13. To save resin for future use, add 10 BV of DI water, discard eluate. Then add 5 BV of charge buffer, discard eluate. Add 10 BV of DI water, discard eluate. Finally, add 5 BV of binding buffer, and drain into waste container until about 3mL remains above resin bed. Store at 4 degrees Celsius. For use next time, drain out buffer until only a little remains above resin bed surface.
SDS PAGE of IMAC Fractions
BCA Assay of IMAC Fractions + Concentrators
Transformation of BBa_I74609 for sfGFP Control
1. Resuspend Spring 2015 Distribution Well 8M in 2015 Kitplate 3 with 10 uL of nuclease free ddH20. 2. Incubate in the well for 5 minutes to absorb residual lyophilized DNA. 3. Mix gently and transfer DNA from the well into a labeled PCR tube.
4. Place one aliquot of DH5alpha chemically competent E. coli on ice to thaw for 10 minutes. 5. Mix 1uL of plasmid in the thawed cells gently. Incubate on ice for 10 minutes. 6. Rescue cells with 400 uL of SOC, and incubate for 1 hour at 37 deg with gentle shaking at 300 rpm. 7. Plate entire mix of cells on prewired agar plates with appropriate ABX. 8. Let plates dry, invert, and place in incubator for 16 hours.