DESCRIPTION

TOROBlue^® One HS KOD Kit  includes 2× One HS KOD Mix with dye is  a  ready-to-use premixed solution containing genetically modified hotstart KOD DNA polymerase, PCR buffer, dNTPs, elongation accelerator and loading dye. This mix which enables fast PCR, which has an extension time of  5 sec/ kb  by applying  genetically modified  KOD DNA polymerase and a new elongation accelerator. In addition, this mix  provides  greater  efficiency  and elongation capabilities than conventional PCR enzymes. In particular, these show greater amplification success from crude specimens.

FEATURES

-Fast: can amplify the targets using the following very short conditions: 0~ 10 kb: 5 sec/ kb ; ≥ 10 kb: 10 sec/ kb

-Easy-to-use: premixed with loading dye for directly loading the gel of Post-Staining or Precasting .

-High Fidelity：exhibits approximately 80-fold higher fidelity than Taq DNA polymerase for cloning.

-Direct PCR: effective for amplification from crude samples.

APPLICATION

-Direct PCR

-Colony PCR

-Amplification of NGS libraries

-Site direct gene mutation

COMPONENTS

The kit includes the following  reagents,  which can be used for 40 reactions for a total 50ul reaction volume, or 100 reactions for a total 20ul reaction volume.

Cat No. ：KAO-201  

2× One HS KOD Mix  with Dye                      1ml ×1 tube

PROTOCOL

1. Standard reaction setup

(1) Before preparing the mixture, all components should be completely thawed on ice.

(2) Gently mix the reaction solutions and spin down in microcentrifuge.  

Notes:

  -Primers should be 22-35 bases with  Tm ≥ 65°C.

  -Optimal primer concentration is 0.3uM. In the case of long targets (≥10 kb), reduced primers concentration (0.15uM) may give more effective amplification.

  -When PCR yield is low, increased primers concentration (0.5 uM) may give more effective amplification.

  -Decreased the amount of template DNA when no PCR Product.   

2. Cycling conditions

Notes:

- Extension time: 0-10kb, 5sec/kb; ≥ 10 kb, 10sec/kb.

-Longer extension time may enhance efficiency. For amplification from a low copy DNA or crude sample, the extension time should be 10 sec. /kb.

-Poor amplification may be improved by changing the denaturation step to 94°C, 15sec.   

3. Electrophoresis 

(1) Directly loading the PCR products and the suitable TOROBlue^® Loading Marker  into the Post-Staining or Precasting agarose gel.

(2) Run on agarose electrophoresis to detect PCR products and marker.

STORAGE  

  This reagent can be stored at 4°C for 1 months. For longer storage,  this reagent should be kept at -20°C.

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