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M. hyopneumoniae PCR

互联网

2093

1 . Because of the high sensitivity of this protocol, it is recommended that the outer PCR run in a single tube format and followed by either single tube or strip format for inner PCR . This is to reduce the possible cross contamination from one tube to the other.

2 . The following solutions need to be prepared:

 

 Tag 5U/µl    
 Primers    
   TH132/TH133 (25 µM )  Outer PCR
   Mhp3/Mhp4 (5.5 µM )  Inner PCR
 dNTPs   2 µM  Inner PCR
 MgCl2  50 mM  Inner PCR
 MasterAmp E  2X  Outer PCR
 Qiagen buffer  10X  Inner PCR
 Q solution  5X  Inner PCR
 SuperQ H2O    
     

Outer PCR :

3.1 There are 29 wells available for running 0.5 ml tubes at one time in a Ericopm TwinBlock thermocycler. Each run should includes the following controls:

Negative control: No DNA
Positive control: 100 fg/10µl
10 fg/10µl
2.5 fg/10µl (or 1 fg/10µl)
1 ng/10µl (or 0.5 fg/10µl)

3.2 This leaves 24 wells for clinical samples. If cross contamination is suspected to be a problem, it is recommend that more negative controls can be every other 5 clinical samples.

 

 25 µM TH132

 25 µM TH133

 MasterAmp E 2X Buffer

 H2O

 5 U/µl Taq

 Template DNA

 Total volume

 2 µl

 2 µl

 25 µl

 11 µl

 0.25 µl

 10 µl

 50 µl

3.3 Master solution should be prepared by mixing all the components except template DNA. Then, 40 µl is dispensed into each tube. The volume of the master solution should be prepared such that it is enough for extra one or two reactions than the number of samples intended to run.

3.4 The reaction includes 4 minutes denaturation at 94°C followed by 30 repeats of denaturation at 94°C for 1 minute, annealing at 48.8°C for 1 minute, and extention at 72°C for 1 minute. The reaction is then incubated at 72°C for 7 minute.

Inner PCR :

4.1 10 µl of outer PCR product is diluted into 990 µl H2O in eppendorf tube. The dilution is mixed by vortex followed by a short spin in a microfuge to make certain that no diluent was attached to the inner ring of the lid of the eppendorf tube, which might result in an increase of cross contamination during opening and closing of the eppendorf tube.

 

 5.5 µM Mhp3

 5.5 µM Mhp4

 10X Qiagen Buffer

 2 mM dNTPs

 H2O

 5X Q Solution

 5U/ µl Tag

 Template DNA

 Total Volume

 1 µl

 1 µl

 5 µl

 1 µl

 30 µl

 10 µl

 0.25 µl

 2 µl

 50 µl

4.2 Master solution should be prepared by mixing all the components except template DNA. Then, 48 µl is dispensed into each tube. The volume of the master solution should be prepared such that it is enough for extra one or two reactions than the number of samples intended to run.

4.3 The reaction includes 2 minutes denaturation at 92°C followed by 30 repeats of denaturation at 92°C for 1 minute, annealing at 56°C for 1 minute, and extention at 72°C for 1 minute. The reaction is then incubated at 72°C for 7 minute prior to analysis in 1 % agarose gel with 10 µl of reaction mixture.


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