Lab 7: LAB ASSIGNMENTS
PRELAB QUIZ
Visit Canvas to take the quiz. The quiz questions are also listed below for your convenience:
Question 1
What experiments are being simulated this week? What is the purpose of those experiments? (Provide 2-3 sentences).
Question 2
What volume of IPTG will you add to each culture?
How many cultures do you need to induce with IPTG?
Given the two answers above, what total volume of IPTG do you need for this experiment?
Question 3
Which buffer will you use to equilibrate, wash, and elute during the column purification?
Buffer 1: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole
Buffer 2: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole
Buffer 3: 50 mM NaH2PO4, 300 mM NaCl
HINT: Thinking about the purpose of each step will help determine which buffer should be used:
- Equilibration: the purpose of this step is to prepare the Ni-NTA resin in a buffer similar to what you will wash and elute with, so that the HCAII will optimally bind
- Wash: the goal of washing is to remove proteins that might have a small affinity for the Nickel resin (such as those with several naturally occurring histidines). Therefore the wash buffer must remove these proteins without removing HCAII.
- Elution: the goal of elution is to remove HCAII from the column so that you can collect it as it flows off.
IN-LAB ACTIVITIES
- After joining BBCU, you and your partner will join a breakout room.
- Your TAs will provide you a link to a Google doc. You and your partner will work together to complete it. It will include:
- An analysis of each SDS-PAGE sample collected throughout the protocol.
- A table where you will outline each step in the protocol, state its purpose, and write down any questions you have. Each pair is required to ask a TA at least one question about the protocol.
- Based on the provided NanoDrop results, you will decide which elution fractions to dialyze.
- Before leaving BBCU, meet with a TA to ask them at least one question about the protocol. They will also discuss your elution fraction choices with you.
- Complete the lab/partner/self evaluation Google form.
- Get a sharable link to your Google doc and submit it on Canvas.