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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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