In this exercise, you will perform a polymerase chain reaction (PCR) to amplify the cloning region of a given series of cloning vectors. The purpose is to help you familiarize yourself with one of the most versatile and important tools in the genetic engineering toolbox. The activity will also provide you with positive controls for future independent experiments, as well as DNA material for the Subcloning of PCR fragments exercise
Learning Objectives
Abilities
At the end of the activity, you will be able to:
Prepare a Master Mix
Measure desired DNA quantities
Run a PCR amplification
Skills
Preparing reagents
Using a PCR machine
Using Benchling to determine primers
Setting up a PCR reactions
Instructions
1. Calculate how much DNA sample you will need to add to your PCR reactions.
2. Use the following table to determine how much of each reagent you will need to add.
Reagent | Quantity |
5x Phusion HF Buffer | 4uL |
10mM dNTPs | 0.5uL |
Phusion DNA Polymerase | 0.25uL |
Forward Primer | 1uL |
Reverse Primer | 1uL |
DNA | ~200ng |
Water | Fill to 20uL |
Total Volume | 20uL |
Our goal is to amplify two distinct fragments - the mRFP from the pSB1-C3 plasmid and the Kan-backbone from the pECIA-Blue plasmid.
3. Retrieve 4 PCR tubes. Label them as the following 1 - mRFP PCR, 2 - mRFP control, 3 - pECIA PCR and 4 - pECIA control. The only difference between the control and the PCR tubes will be the lack of your DNA in the controls (instead you will add more water). Ensure that you add the primers that correspond to the correct parts for the mRFP and the pECIA tubes.
4. Pipette all elements into each PCR tube
5. Determine what the time frame for the PCR reactions will be and place the tubes into the thermocycler.
​ | Step | Temp | Time | Notes |
​ | Initial Denaturation | 98°C | 30seconds | ​ |
Repeat for 25-35 Cycles | Denaturation | 98°C | 5-10seconds | ​ |
Repeat for 25-35 Cycles | Annealing | 45-72°C | 10-30 seconds | Temperature is primer dependent (calculate average melting point of both primers and subtract 5) |
Repeat for 25-35 Cycles | Extension | 72°C | 15-30sec/kb | Time depends on size of fragments (find the size on Benchling by highlighting the area you are amplifying) |
​ | Final Extension | 72°C | 5-10min | ​ |
​ | Hold | 4°C | Infinite | ​ |
6. Once the thermocycler is done run your PCR products on a agarose gel. For this retrieve new PCR tubes and add 5uL of your PCR product to these new tubes. Add 1uL of loading dye to the lid of the tubes. Close the tubes and centrifuge to pull the dye through the solution. You can now add 6uL of your sample to your gel.
7. If your gel shows the correct bands for your PCRs then you may proceed to the PCR purification step (Protocol 8).
A visual on how to conduct a Polymerase Chain Reaction (PCR). Note that there might be some slight variation between our protocol and the Addgene protocol.
