Superstart Taq DNA Polymerase M3 HYA117
Superstart Taq DNA Polymerase M3 is an antibody-mediated hot start third-generation mutant of Taq, specifically designed for fast PCR and resistance to common PCR inhibitors such as those found in human samples (blood, sputum, urine, and stool), as well as carryover inhibitors from sample preparation.
1. The enzyme is stable at 15- 25°C for several hours, depending on the composition of the final working solution, and is therefore suited for use in automated processes.
2. Superstart Taq DNA Polymerase M3 retains 5′→3′ exonuclease activity, enabling probe- or dye-based qPCR for both routine and challenging sample types.
3. The glycerol-free formulation makes Superstart Taq DNA Polymerase M3 suitable for preparation of dried amplification mixes.
Components
Superstart Taq DNA Polymerase M3 (Glycerol-free), 30 U/µL
Storage
Store at -15~ -25°C.Avoid repeated freezing and thawing (more than 5 times).
Instruction
1.Reaction system
|
Components |
Volume |
Concentration in Master Mix |
|
2× PCR Buffer including MgCl2* |
10 uL |
1× MgCl2 3-4.5 mM |
|
20× Primer Mix** |
1 μL |
1× |
|
20× Probe Mix*** |
1 μL |
1× |
|
Superstart Taq DNA Polymerase M3 |
1 μL |
0.02 U or 0.04 U/µL PCR |
|
DNA template |
5 μL |
/ |
|
ddH2O |
2 µL |
/ |
|
Total |
20 uL |
/ |
*Suitable concentrations of primers range from 200 to 500 nM (final concentration). The primer concentration may be optimized by varying the final concentration from 200 to 500 nM in increments of 100 nM.
**Suitable concentrations of probes such as hydrolysis probes range from 50 to 500 nM (final concentration).The probe concentration may be optimized by varying the final concentration from 50 to 500 nM in increments of 100 nM.
***Suitable MgCl2 concentration ranges from 3 to 4.5 mM. A higher MgCl2 concentration leads to a better inhibitor tolerance.
2. Reaction Program
|
Step |
Temperature |
Time |
Cycles |
|
Pre-Denaturation |
98 ℃ |
30s-5min* |
1 |
|
Denaturation |
95 ℃ |
1s-1min |
45** |
|
Annealing-Extension |
50~65℃*** |
1s-1min |
|
|
Cooling |
40 ℃ |
10 s |
1 |
*Use Pre-Denaturation of 3 minutes at 98°C to ensure full denaturation of complex target DNA. A Pre-Denaturation time of up to 10 minutes may be required for some crude samples.
**For most assays, 45 cycles are suitable. If the assay shows steep amplification curves and early crossing points, 40 cycles should be sufficient. Reducing the number of cycles will improve uniformity of the product and reduce the time required for the assay.
***Depending on the annealing temperature of the primer. A 10s annealing/extension/data acquisition time may be used with most assays; increase to 30 s/kb up to 1 min/kb for difficult amplicons or templates. This must be determined empirically. For short amplicons ≤100 bp, a denaturation and extension time of 1s might be favorable.
Application
For fast and inhibitor-resistant PCR. Formulated without glycerol for the preparation of dried amplification mixes.
Notes
1.Use nuclease-free techniques; nuclease-contaminated reagents and reaction vessels may degrade template DNA.
2.Wear disposable gloves and change them frequently.
3.Avoid touching surfaces or materials that could cause nuclease carryover.
4.Clean and decontaminate work areas and instruments, including pipettes, with commercially available decontamination reagents.
5.Use only new nuclease-free aerosol-blocking pipette tips.
6.Use a work area specifically designated for nucleic acid work, and if possible, use reaction vessels and pipettes dedicated only for work with template nucleic acid.