DESCRIPTION

    TOROGreen^® One HS KOD Kit includes two components，2×One HS KOD Mix and TOROGreen^® 6×DNA LoadingBuffer. 2× One HS KOD Mix is a ready-to-use premixed solution containing genetically modified hotstart KOD DNA polymerase, PCR buffer, dNTPs, elongation accelerator. 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. TOROGreen^® 6×DNA loading buffer contains the fluorescence dye, which is directly detected on 470nm LED transilluminator after the DNA electrophoresis.

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: offer TOROGreen^® 6×DNA loading buffer premixed with fluorescent dye for directly observed on 470nm LED transilluminator after electrophoresis.

  -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-101  

2× One HS KOD Mix                        1ml ×1 tube

TOROGreen^® 6×DNA loading buffer          1ml ×1 tube

We also offer more small package as the following.

Cat No. ：KAO-100S      

2× One HS KOD Mix                        0.25ml ×1 tube

TOROGreen^® 6×DNA loading buffer          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) Add 1 volume of TOROGreen^® 6×DNA loading buffer to 5 volumes of PCR products.

   (2) Mix well, spin down and load the mixture and the suitable TOROGreen^® Loading Marker.

   (3) Run on agarose electrophoresis to detect PCR products and marker. No additional dye is required for the PCR products and marker.

   (4) Realtime observation of electrophoresis is also possible if a 470nm blue light source is fitted to the electrophoresis tank.

   (5) Use the 470nm blue-light transilluminator or imaging systems photograph the gel.

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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