Lab 2: PROCEDURE Part 3
PROCEDURE: PCR
REMINDERS:
Each student is completing this experiment individually.
For the insert reaction, everyone is amplifying the wild type HCAII gene. The teaching staff will be making all the mutants.
Begin by calculating the volume of all of the components for the three different reactions you will be running.
This quick video explains how to do the dilution calculations for PCR, and includes some practice problems:
The table below lists each reagent needed for PCR, the stock solutions that you will be provided, and the desired final reaction conditions:
| Reagent | Stock Solutions Provided | Desired Final Reaction Conditions |
| Buffer | 5x | 1x |
| dNTP mix | 10 mM (includes 10 mM of each nucleotide) | 200 μM |
| Template | 2.5 ng/μL (actual concentration will be provided in lab) | 10 ng |
| Forward and reverse primers | 10 μM | 400 nM |
| Phusion polymerase | 1 U/μL (A “U” or “unit” is a standard way of measuring an amount of enzyme in a solution, and relates to its activity) | 1 U |
| Sterile water | To a final volume of 50 μL |
Use the information above to create a table in your lab notebook that looks like the one below. You should arrive to lab with as much of the table completed as possible.
Remember that you will be setting up three separate PCRs:
Reaction #1: HCAII template, insert primers
Reaction #2: pETblue2 template, vector primers
Reaction #3: pETblue2 template, vector primers, no Phusion (negative control)
| Reagent | Reaction 1: Insert | Reaction 2: Vector | Reaction 3: negative control |
| 5X reaction buffer | |||
| dNTP mix | |||
| Insert primers | |||
| Vector primers | |||
| DNA template | |||
| H20 | |||
| Phusion | |||
| Total Volume | 50 μL | 50 μL | 50 μL |
Once you are confident in your calculations, get your aliquots from the back bench. Keep the aliquots and your PCRs on ice until you put them into the thermocycler. This will help maintain the activity of your Phusion enzyme.
Prepare the reactions, per your calculations, into PCR tubes. It is important that you label the PCR tubes clearly and in a way that will allow you to distinguish your reactions from your classmates’. The teaching staff will take the PCRs out of the machines the next morning, and all the tubes will be placed into one large freezer box.
Be sure to add Phusion to the reaction last. Remember how to pipette small volumes! Once all the components have been added to the tubes, mix them with a pipette or by gently flicking the tubes. Do not vortex! Centrifuge briefly in the nanofuge to be sure the samples are all at the bottom of the tubes. You can spin the tiny PCR tubes by placing them inside a 1.5 mL eppendorf tube (people sometimes break the caps off the eppendorf tubes and save them as centrifuge adaptors).
Programming the thermocycler
Once you have finished setting up your PCRs, program the thermocycler to run the following program (also refer back to Figure 2.4):
Step 1: 98 °C for 30 seconds
Step 2: 98 °C for 10 seconds
Step 3: 50 °C for 30 seconds
Step 4: 72 °C for 2 mins
Repeat steps 2–4 for 35 cycles
Step 5: 72 °C for 10 mins
Step 6: 10 °C hold
Detailed instructions for programming the thermocycler can be found in the bench drawer or next to the PCR thermocycler. If you have questions, please ask your TA.
Keep the reactions on ice while programming the thermocycler.
You need about four people per thermocycler, so don’t start the program until at least four people are ready.
Clean up
Empty tip jar to the “USED TIPS” box (note that the box is for used tips only; all other non-sharps, such as gloves, should go to regular trash).
Dispose of or put back reagents (see blackboard for details).
Dispose of 96-well plate in regular trash.
Leave check-in sheet on the bench.
Put everything from the drawers back to the corresponding drawers. If supplies (such as beakers) are used to hold liquid, wash the supplies and leave them on the bench to dry. Do NOT dry them on the drying rack above the sink.