PCR amplification of plasmid DNA (Phusion)
Updated 8/3/2019 07:43 PM
Introduction
Materials
- Labware
- Thin walled 200µL PCR tubes
- Racks capable of holding PCR and microcentrifuge tubes
- Sterile pipette tips
- Permanent Marker
- Equipment
- Pipettes for pipetting volume ranges from 0.25 μL to 50 μL
- Microcentrifuge capable of holding 1.5mL tubes and producing at least 17,900 x g
- Thermocycler
- Reagents
- PCR Reagents as described below in Table 2.
Procedure
Important information to be documented throughout this procedure
Table 1: Check to make sure you have all information
A A | B B | |
1 1 | Information | Check |
2 2 | DNA plasmid template used in PCR reaction and concentration of sample | |
3 3 | Expected fragment size | |
4 4 | Primers and DNA polymerase used in the reaction | |
5 5 | Volumes used in PCR reaction | |
6 6 | Controls used in PCR reaction | |
7 7 | Thermocycler used | |
8 8 | Amplification program used | |
9 9 | Samples’ names, dates and storage location | |
10 10 | Unexpected and expected observations and results (either in writing or in picture form) | |
11 11 | Deviations/Alterations of the protocol | |
12 12 | Clarification notes (issues, significant notes) |
PCR with Phusion Polymerase
- Start by filling in the table of reagents that will be added to the reaction mixture as below:
Table 2: Reagents for a Phusion PCR reaction
A A | B B | C C | D D | E E | |
1 1 | Reagent | Location | Concentration of stock solutions | Volume | Final concentration |
2 2 | Sterile water | -20 °C freezer | To 20 µL or 50 µL | ||
3 3 | PCR buffer: Phusion HF buffer | -20 °C freezer | 5X | 1X | |
4 4 | dNTPs | -20 °C freezer | 10 mM | 200 μM | |
5 5 | MgCl2 | -20 °C freezer | In buffer | 1.5 mM | |
6 6 | Forward primer: ______ | -20 °C freezer | 10 μM | 0.5 µM | |
7 7 | Reverse primer: _____ | -20 °C freezer | 10 μM | 0.5 µM | |
8 8 | Template DNA: _____ | -20 °C freezer | Variable | 10 pg - 250 ng | |
9 9 | Phusion DNA polymerase | -20 °C freezer | 5 Units/μl* | 1 unit/50uL rxn✝ | |
10 10 | Final volume | 25 µL or 50uL | |||
11 11 | ✝Final concentration of polymerase may vary between manufacturers | *Units may vary between manufacturers | |||
- Arrange all reagents needed in a cold block (except Taq DNA polymerase) and let them thaw completely before setting up a reaction. Gently vortex and briefly centrifuge all solutions after thawing.
CRITICALKeep the reagents on ice throughout the experiment.
- Label the required PCR tubes with permanent marker and include sample details or code, as in the example of table 2 above.
- Pipet the reagents following the order in table 2:
Note: MgCl2 may be already in the buffer, verify the required final concentration for your experiment.
Note: Be careful to avoid contamination of your reagents, and DNA sources. When working with DNA and enzymes, it is highly recommended to not reuse tips.
- In a separate PCR tube add all the reagents with the exception of template DNA for a negative control (increase the water to compensate for the missing volume).
- When needed in your experiment, prepare in the same manner, a positive control reaction with template of known size and appropriate primers.
- Gently mix the solution by “finger vortexing” (flick with finger several times), to allow dispersal of Taq DNA polymerase in the reaction mix.
Note: Care should be taken to avoid the formation of bubbles.
- Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture
- Place the PCR tubes into the selected thermocycler.
- Once the lid to the thermocycler is properly closed, start the required amplification program, as in table 3.
Table 3: Phusion Amplification protocol
A A | B B | C C | D D | |
1 1 | Cycle step | Cycling temperature | Time | Number of cycles |
2 2 | Lid Temperature | 2°C higher than highest temperature | ||
3 3 | Initial denaturation | 98 °C | 30 s | 1 |
4 4 | Denaturation | 98 °C | 10 s | 30 |
5 5 | Annealing | 45-72 °C | 30 s | 30 |
6 6 | Extension | 72 °C | 30 s* | 30 |
7 7 | Final Extension | 72 °C | 5 min | 1 |
8 8 | Hold | 4 °C | ∞ | 1 |
9 9 | Note: protocol may differ if using different DNA polymerases. | |||
10 10 | ||||
- If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20°C for long-term storage in assigned storage box.
- Properly record the storage location in the corresponding section in Benchling.
This protocol describes the method used for the amplification of specific DNA fragments by the Polymerase Chain Reaction, using plasmid DNA as a template. The amplified products may subsequently be analysed for presence or absence and size in agarose gels. Following purification, these can be used in downstream assays, including sequencing, ligation and transformation and restriction digestion.
An extensive SOP is located here:
SOP: PCR amplification of plasmid DNA It is recommended to read at least Section 7 - Important Notes.