Description
Chai’s 2X PCR Master Mix is an optimized ready-to-use solution for routine PCR and qPCR. The mix uses an open Taq polymerase in conjunction with enhancers and stabilizers to deliver exceptional performance at low cost. The Master Mix is compatible with dye and probe based assays, and products between 100 bp and 5 kb can be amplified.
Specifications
| Concentration | 2X |
| Storage Temperature | -20 °C |
| Exonuclease activity | 5' → 3' |
| Hot Start | No |
| Recommended reaction size | 10 - 50 µL |
Composition
| Component | Amount |
|---|---|
| Taq polymerase | 50 U/mL |
| KCl | 100 mM |
| MgCl2 | 6 mM |
| TrisCl pH 8.6 | 20 mM |
| Glycerol | 10% |
| Trehalose | 200 mM |
| BSA | 0.4 mg/mL |
| Detergents | 0.26% |
| dNTPs (each) | 600 µM |
Performance
Lambda Phage Standard Curve Amplified with Chai PCR Master Mix 2X
Lambda Phage Gel Electrophoresis
Protocol
Thaw the 2X PCR Master Mix at room temperature.Vortex the Master Mix gently, and spin it briefly in a micro-centrifuge to collect the material in the bottom of the tube.
Reaction Set Up:
Assemble reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (95 ºC). The recommended reaction volume is 25 µL. Reaction volumes between 10 and 50 µL may be used; scale up/down the reaction components accordingly. Final concentration of the Master Mix in the reaction should be 1X.
| Component | 25 µL REACTION VOLUME |
|---|---|
| 10 µM Forward Primer | 0.5 µL |
| 10 µM Reverse Primer | 0.5 µL |
| Template DNA | 1 ng - 1 µg Genomic |
| 0.5 pg - 5 ng Plasmid / Viral | |
| 1 ng - 100 ng cDNA | |
| 2X Master Mix | 12.5 µL |
| Nuclease-Free Water | Bring up to 25 µL |
THERMOCYCLING CONDITIONS
Three-Step PCR
| Step | Temp | Time |
|---|---|---|
| Initial denaturation | 95 °C | 30 s - 2 min |
| Denature | 95 °C | 15 - 30 s |
| Anneal | 45 - 68 °C | 15 - 30 s |
| Extend | 68 °C | 1 min/kb |
| Final Extension | 68 °C | 5 min |
| Hold | 4 °C |
Cycle 30 - 40x
Two-Step PCR
When primer annealing temperatures are above 60 °C, a two-step PCR can be used.
| Step | Temp | Time |
|---|---|---|
| Initial denaturation | 95 °C | 30 s - 2 min |
| Denature | 95 °C | 15 - 30 s |
| Anneal/Extension | 45 - 68 °C | 1 min/kb |
| Final Extension | 68 °C | 5 min |
| Hold | 4 °C |
Cycle 30 - 40x
