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      1. 核酸檢測服務

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        PCR labeling DIG Probe kit

        Cat number: 001.1145

        Size:25T

        Storge:-20

        The labeled probe application

        FISH, ISH,

        Southern blotting, Northern blotting,

        Bacteria ISH.

         

        1.      Introduction:

        PCR labeling DIG Probe kit is one rapid method for preparing DIG-labeled Probe. When the template is available in not too long (<2kb). It requires no optimization than other methods and produces a high yield of DIG probe. In PCR labeling, DIG-dUTP was add into PCR specific region of the template DNA. The result is a highly labeled, very specific, and sensitive hybridization probe.

         

        2.PCR DIG Probe Synthesis Kit

        1) Enzyme mix,

        2) PCR DIG probe synthesis mix, 10×.

        3) PCR buffer with MgCl2, 10×. [1]

        4) dNTP stock solution, 10×. [1]

         

        3.Prepare DNA and primers:

        (1)DNA 50ng

        (2)Forwards primer:0.5uM solution

        (3) Reveres primer:0.5uM solution

         

        4. Prepare PCR reaction system:

        PCR buffer with MgCl2, 10×:       5ul

        PCR DIG mix,10×.                  5ul

        Forwards primer:                    1ul

        Reveres primer:                     1ul

        Enzyme mix:                          1ul

        Template DNA: (10-20ng)   1-2ul

        Sterile H2O:        Add up to  50ul

        Mix 1 min

        5.Set up PCR system:

        1) Initial denaturation: 95°C 3 min –

        2) 95°C 30 s, 60°C 30 s, 72°C 40 s

        Cycles:1 – 10

        95°C 30 s, 60°C 30 s, 72°C 40 s

        Cycles: 11 – 30

        Final elongation 72°C 7 min

         

        6. Next application:

        1) Use 5-8 μl of the reaction mixture to check the results of the labeling reaction.

        2) PCR productions are stored at 4°C.

        3) PCR-generated probes can be used directly in the hybridization reaction. It not require clean up.

        4)  2 μl/ml in hybridization solution is suitable.

         




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