The following case studies will allow you to practice everything you've learned in the previous units. If you need a refresher, use the links below to revisit the information:
You are preparing to probe a membrane as part of a Western blot protocol. To do so, you’ll need to:
Let's get started!
You plan to block your membrane in 25 mL of 5% (w/v) milk in TBST buffer.
When weighing the powdered milk, you accidentally weighed out 10 g and added it to your 25 ml of buffer. How many additional mL of TBST do you need to add to this solution to create a 5% w/v solution? mL Note: report value to the nearest whole number.
You need 10 mL of 1:160000 diluted primary antibody solution to incubate your membrane.
True or false: This concentration could be created directly with standard pipettes.
A reasonable serial dilution concentration would be
To create 10 mL of the 1:160000 primary antibody solution, add μl of the serial dilution to ml of buffer. Note: report values to the nearest tenth.
Your secondary antibody comes lyophilized, and the datasheet says that 5 mg are provided. You plan to use it at 1.25 μg/mL for a 10 mL incubation.
How many mg of the lyophilized antibody would you add to 10ml to achieve the proper working concentration? mg
You add 1 ml of buffer to the antibody to create a 5mg/ml dilution.
What is the w/v concentration of this dilution? % w/v.
If the molecular weight of a secondary antibody is estimated at 155,000 g/mol, what is the concentration of this solution in micromolarity? μM Note: report values to the nearest tenth.
To create 10 ml of the 1.25 μg/mL working solution from the 5 mg/ml dilution, the following solution should be made:
μl of the 5 mg/ml dilution Note: report values to the nearest tenth.
ml of the buffer Note: report values to the nearest tenth.
You're preparing to set up a PCR reaction and run your product on a gel. To do so, you'll need to:
Let's get started!
You ordered two 20-nucleotide primers and received 41 nanomoles of each as lyophilized powder. Note: nano is a metric unit; there are 1000nano- in 1μ-.
Your lab manager tells you to reconstitute each primer to a concentration of 100 μM. How many μl of TE buffer should you add to each primer to achieve this concentration? μl
You talk to a different person in the lab and they tell you to make a 0.1% w/v dilution. Assuming an average molecular weight of 330 g/mol per nucleotide, how many μl of TE buffer should you add to each primer to achieve this concentration? μl of TE buffer. Note: report values to the nearest whole number.
You aren't sure what to do so you add 1000 μl of TE buffer to the lyophilized primers. What is the concentration (in μM) that you have created? μM
After reviewing the PCR protocol, you see that you need to add 1 μL of each 10 μM primer to the PCR reaction (25 μl total reaction). For the sake of simplicity, we will focus on just one of the primers in the next exercises.
You decide to create 10 μl of the 10 μM solution for the forward primer. To create this solution, you would mix the following:
μl of your 41 μM solution Note: report values to the nearest tenth.
μl of buffer
What is the final concentration (in μM) of the forward primer in the 25 μl PCR reaction? μM
After running your PCR, you want to confirm the size of the amplified regions. You decide to pour a 0.8% agarose gel for electrophoresis.
How many grams of agarose should you weigh out to add to 50 mL of TAE buffer? g
Your lab manager sees that you are pouring a gel and informs you that the expected size of the PCR product is quite small so you should run a 1.8% agarose gel. Luckily you haven't poured your gel yet. How many additional grams of agarose do you need to add to your agarose-buffer solution to create a 1.8% agarose gel? g
Did you notice a typo, an incorrect answer, or another aberration? Send an email here to let us know.