Jan 10, 2022
Version 3
General Taq PCR Master Mix -- CHEM 384/584 V.3
- 1Brigham Young University
Protocol Citation: Ken Christensen 2022. General Taq PCR Master Mix -- CHEM 384/584. protocols.io https://dx.doi.org/10.17504/protocols.io.b3npqmdnVersion created by Ken Christensen
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: January 10, 2022
Last Modified: January 10, 2022
Protocol Integer ID: 56751
Abstract
2X PCR Master Mixes are convenient to use as they include all the necessary PCR components except the template and primers. Most PCR Master Mixes also include agarose gel running dyes and a density reagent that allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR.
Guidelines
Storage: -20℃ for long-term storage. 4℃ for short-term storage (up to 3 months). (Note) If used frequently, store at 4℃ ; the activity of the Master Mix may decrease with repeated freezing and thawing. Gently mix well before use and centrifuge briefly.
Application:
・DNA amplification by PCR
・Colony PCR
PCR Products: Since most PCR products amplified Taq PCR Master Mix have an A overhang added at 3’-termini, the obtained PCR product can be used directly for cloning into a TA cloning vector. Additionally, it is possible to clone the product in a blunt-end vector after blunting and phosphorylation.
Dye Migration during Electrophoresis: Check individual manufacturers information on the migration of the included running dyes.
Setup Reaction
Setup Reaction
To a 25 µL aliquot of a 2X Taq PCR Master Mix (e.g. TaqDog, or Sapphire Amp), add template (10-20 µl cleared lysate for colony PCR or 20-50 ng of purified DNA for typical PCR), forward and reverse primers to a final concentration of 200 nanomolar (nM) . Adjust final volume to 50 µL with nuclease free water or autoclaved water.
| A | B | |
| 2X Master Mix | 25 ul (pre-aliquoted and stored in the freezer) | |
| Template | 10-20 ul of bacterial lysate or 20-50 ng purified DNA | |
| Forward Primer | 1 ul of 10 uM primer dilution | |
| Reverse Primer | 1 ul of 10 uM primer dilution | |
| ddH2O | to a final volume of 50 ul |
Run Reaction
Run Reaction
followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec.
| A | B | C | |
| Initial denature | 94C | 1 minute | |
| Denature | 94C | 30 seconds | |
| Anneal | 55C | 30 seconds | |
| Extension | 72C | 1 min/kb | |
| Repeat steps 2-4 | 25-40x | ||
| Final extension | 72 | minutes | |
| Cool | 4C | Until cancelled |
A typical thermocycling program for a PCR for amplicons less than 1 kb is 1 minute. For longer amplicons, adjust the program to 1 minute/kb seconds for the extension and final extension times.