Difference between revisions of "20.109(S17):In silico cloning and induction of protein expression (Day1)"
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==Protocols== | ==Protocols== | ||
| − | ===Part 1: Subculture BL21(DE3) pRSETb_FKBP12 cells=== | + | ===Part 1: Laboratory orientation quiz=== |
| + | Complete the orientation quiz with your partner. Though you are working with your partner, each student should record all answers on the provided quiz. If you disagree with your partner on an answer, you should write what you think is the correct answer on your quiz. | ||
| + | |||
| + | Good luck! | ||
| + | |||
| + | ===Part 2: 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. | 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. | ||
#Acquire a 125 mL flask from the front laboratory bench and add 50 mL of LB to the flask. | #Acquire a 125 mL flask from the front laboratory bench and add 50 mL of LB to the flask. | ||
| Line 13: | Line 18: | ||
#Subculture the overnight culture in the 50 mL LB such that the OD<sub>600</sub> = 0.2. | #Subculture the overnight culture in the 50 mL LB such that the OD<sub>600</sub> = 0.2. | ||
#Move the flask to the 25 °C water bath shaker. | #Move the flask to the 25 °C water bath shaker. | ||
| − | #Check the OD<sub>600</sub> of your culture every hour and when the OD<sub>600</sub> = 0.8, complete Part | + | #Check the OD<sub>600</sub> of your culture every hour and when the OD<sub>600</sub> = 0.8, complete Part 4. |
| − | ===Part | + | ===Part 3: 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. | 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 | + | ===Part 4: Induce FKBP12 protein expression=== |
#Add IPTG to a final concentration of 1 mM and return the flask to the 25 °C water bath shaker. | #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. | 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. | ||
Revision as of 20:14, 3 January 2017
Contents
Introduction
Protocols
Part 1: Laboratory orientation quiz
Complete the orientation quiz with your partner. Though you are working with your partner, each student should record all answers on the provided quiz. If you disagree with your partner on an answer, you should write what you think is the correct answer on your quiz.
Good luck!
Part 2: 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.
- Acquire a 125 mL flask from the front laboratory bench and add 50 mL of LB to the flask.
- 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.
- Subculture the overnight culture in the 50 mL LB such that the OD600 = 0.2.
- Move the flask to the 25 °C water bath shaker.
- Check the OD600 of your culture every hour and when the OD600 = 0.8, complete Part 4.
Part 3: 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 4: Induce FKBP12 protein expression
- 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
Next day: Purification of induced protein
Previous day: Orientation