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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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