DoGene::Products::PCR Products

Hotstar Taq Polymerase

For research use only.

Description
Hotstar Taq DNA Polymerase is a chemically modified Taq DNA polymerase with stringent hot-start capability. It enables sensitive detection of DNA and effectively reduces primer dimer / non-specific amplifications.

Applications
* Amplification of DNA,identity
* qPCR
* site-directed mutagenesis

Protocol

Primers design and concentration
i. To use appropriate software
ii. Tm: 60°C or higher
iii. To avoid stable secondary structures
iv. To avoid more than 3 consecutive Gs
v. To avoid complementary 3’ ends
vi. 17 ~ 30 nucleotide in length
vii. 200 ~ 400nM each primer

Buffer: the supplied buffer is 10X and contains 15mM Mg2+.
[Mg2+]: although 1.5mM Mg2+ works well for most amplification, the optimal [Mg2+] is determined empirically. Be aware of any chelators in samples.

Amount of Hotstar Taq to be used: start with 2U per 50ul reaction and adjust it by 0.5U increments when it is necessary.

[dNTP]: 0.2mM each dNTP is recommended. [Mg2+] should be adjusted proportionally when [dNTP] is changed.

Thermocycling conditions
1. Activation: 93~95°C, 10min
2. Denature: 93~95°C, 15~30sec
3. Annealing: 60 °C, 30sec
4. Extension: 68 °C, TBD*
5. Cycle number: 30 ~ 40 cycles. It is dependent on starting copy number and length of target.
6. Final incubation: 68 °C, 10min

TBD*: Time is determined by length and sequence of target to be amplified. The rule of thumb is 1min for each kilobases.