20.109(S17):In silico cloning and induction of protein expression (Day1)

Introduction

Protocols

Part 1: Subculture BL21(DE3) pRSETb_FKBP12 cells

Yesterday afternoon, the teaching faculty inoculated 5 mL of LB media with BL21(DE3) pRSETb_FKBP12 and incubated the culture overnight in the 37 °C inucbator. You will use this culture to prepare your sample for the protein purification protocol.

1. Acquire a 125 mL flask from the front laboratory bench and add 50 mL of LB to the flask.
2. Measure the OD₆₀₀ of the overnight culture.
   • Note: You will need to dilute the overnight culture 1:10 to obtain a reading within the range of the spectrophotometer.
3. Subculture the overnight culture in the 50 mL LB such that the OD₆₀₀ = 0.2.
4. Move the flask to the 25 °C water bath shaker.
5. Check the OD₆₀₀ of your culture every hour and when the OD₆₀₀ = 0.8, complete Part 3.

Part 2: Clone pRSETb_FKBP12 in silico

For timing reasons, you will be provided with the pRSETb_FKBP12 product that was cloned by the teaching faculty. In this exercise you will virtually work through the cloning steps that were used to ligate the FKBP12 gene into the pRSETb expression vector.

Part 3: Induce FKBP12 protein expression

1. Add IPTG to a final concentration of 1 mM and return the flask to the 25 °C water bath shaker.

Your culture will incubate for ~16 hr in the water bath then the teaching faculty will collect the cells by centrifugation at XX g for XX min. The harvested cells will be stored in the -80 °C freezer.

Reagents

Navigation links

Next day:

Previous day: