Lab 5: PRELAB
PRELAB BENCH WORK
This week, you are required to come in to lab before your lab period. You need to come in to count your colonies (this may be done any time the lab is open, starting the day after your lab) and to start overnight cultures. Monday lab will need to come in the week before (since the lab is not open on Fridays to Sundays).
Starting overnight cultures
Take a look at your plates and identify the one with the most colonies that are potentially positive clones. This is the plate you will use. If you have multiple plates with lots of good colonies, just pick one of them to use. If you don’t have any colonies, cultures will be prepared for you.
This video demonstrates the procedure for picking colonies and starting overnight cultures:
- Label two test tubes with your name
- Add 2 mL LB with 50μg/mL ampicillin to each test tube (this step has been done for you)
- With a sterile toothpick, touch a single bacterial colony and drop the toothpick into 2mLs
- Repeat for a second time; i.e. each test tube will contain a single colony
- Incubate at 37°C shaking overnight (will be done by the teaching team the day before your lab)
PRELAB ASSIGNMENT
- What will you be doing in lab and why?
- What should you end up with at the end of the lab period? Where will it be located?
- You will encounter a chemical hazard this week in lab. Describe the chemical, the potential risks, and steps you will take to protect yourself.
- Decide which enzyme you will use for your digest screen (see choices below). What size band(s) to you expect if your cloning was successful? What size band(s) do you expect if the cloning was unsuccessful and your colony only contains pETblue2 without HCAII?
Cut sites for each enzyme in both the positive and negative clones are shown here. Note that you will NEED to reference these vector maps to solve this problem!
Remember that the way we designed our cloning experiment actually removed some of the vector sequence (between the dashed lines) in the positive clone.
Remember that you want to choose the enzyme that will give the most clear difference between a positive and negative clone.
See lecture for more information on digest screens!
PRELAB QUIZ
You can take the quiz in Canvas here (link opens in a new tab): Prelab 5 Quiz
In this lab you are screening colonies to see if you have successfully cloned HCAII into pETblue2. You will use an agarose gel to visualize the digested DNA. Which is the single best enzyme to use for this digest screen?
- PstI
- EcoRV
- AlwNI