Evolving Enzymes  

   Inovating IVD

Evolving Enzymes  

   Inovating IVD

MORE
PRODUCT CENTER
TOROGreen® Animal Tissue Direct PCR Kit
    Publish time 2019-09-24 17:21    

TOROGreen® Animal Tissue Direct PCR Kit is is designed to amplify DNA directly from  a wide variety of tissues obtained from mice, human, fish, birds and insects. No DNA purification is required prior to PCR. The kit is based on engineered hotstart KOD DNA Polymerase, a extremely robust PCR enzyme,which can tolerant of many PCR inhibitors present in animal tissue like collagen, melanin and eumelanin (hair, skin) or myoglobin (muscle) and others. TORO Green® 6×DNA loading buffer contains the fluorescence dye, which is directly detected on 470nm LED transillum inator after the DNA electrophoresis. 

DESCRIPTION

TOROGreen® Animal Tissue Direct PCR Kit is is designed to amplify DNA directly from  a wide variety of tissues obtained from mice, human, fish, birds and insects. No DNA purification is required prior to PCR. The kit is based on engineered hotstart KOD DNA Polymerase, a extremely robust PCR enzyme,which can tolerant of many PCR inhibitors present in animal tissue like collagen, melanin and eumelanin (hair, skin) or myoglobin (muscle) and others. TORO Green® 6×DNA loading buffer contains the fluorescence dye, which is directly detected on 470nm LED transillum inator after the DNA electrophoresis. 

FEATURES

-Easy-to-usesample is added directly to PCR reaction, therefore there is no need for time-consuming and expensive DNA purification steps.
-Simple Lysis protocol available—allows more PCR reactions and allows retesting.

-High performance: can amplify 40kb genomic DNA and 90% GC-rich target with 80-fold higher fidelity than Taq DNA polymerase. 

APPLICATION

- Transgene detection

- Genotyping
- Knockout analysis
- Sequencing

COMPONENTS

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

   Cat No. :DPK-200

   Lysis Buffer A                                                         10ml×2tube

   Lysis Buffer B                                                        1.2ml×2tube 

   2× ASFastTM Direct PCR Mix                               1ml ×1tube

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

 PROTOCOL

1. Sample Handling

Direct PCR of Animal tissue

Use a sterile scalpel to cut a very small piece of tissue(0.3-0.5 mm). Place the sample directly into the liquid of the PCR reaction (50μL of volume). Make sure that you see the sample in the solution.

Direct PCR of animal tissue lysis

a. Thermo lysis protocol

-Use a sterile scalpel to cut a very small piece of tissue(about 3mm),such as mouse tail. Then place it into 180μL of Lysis Buffer A.

- Briefly vortex the tube and incubate the sample at 95for 15min in dry bath.

- Add 20ul of Lysis Buffer B and briefly vortex the tube.

- Centrifuge at 12000rpm for 5 min.

- Use 0.5-3μL of the supernatant as a template in a 20μL PCR reaction.

b. Homogenize lysis protocol

-Use a sterile scalpel to cut a piece of tissue(about 50-100mg)and place it in the tube with zirconia crystal beads.

-Add 540μL of  Lysis Buffer A and 60μL of Lysis Buffer B into the tube.

-Fully homogenize the sample according the homogenizer instruction book.

- Use 0.5-3μL of the supernatant as a template in a 20μL PCR reaction.


 2.Standard reaction setup.


1Prepare the mixture on ice as the following components.

 

 


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 (0.5uM) may give more effective amplification.


3.Set up cycling conditions


 

Notes:

-Poor amplification may be improved by changing the denaturation step to 95°C, 15sec.


4.Electrophoresis.


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

-Mix well, spin down and load 5ul of the mixture and 2ul of the suitable TOROGreen® Loading Marker.

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


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



Ordering Information


Cat.No.


Product Name


Size


Store at


Price


Data Sheet


DPK-200


TOROGreen®Animal Tissue Direct PCR Kit 


20μL×100 Reactions

 

-20°C


US$     100.00



 


HOW TO BUY PRODUCTS