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PCR reaction used to verify the strains are bla-NDM positive
Could you provide information on the PCR reaction (enzyme, cycling, primers) that was used to verify the strains are bla-NDM positive?

The protocols that we used were provided by the CDC.

Primers

  1. NDM forward (NDM-F): ATCACCGAGATTGCCGAGC
  2. NDM reverse (NDM-R): GGTTTCGGGGCAGTCGC

 

Master mix set up for one reaction:

a)      5 µL 5X PCR Buffer E (contains 7.5 mM MgCl2, pH 9.0)

b)      14 µL PCR Water

c)       2.5  µL dNTP, 2.5 mM

d)      µL tcdA-F primer, 20 µM

e)      µL tcdA-R primer, 20 µM

f)       0.5 µL Platinum® Taq DNA Polymerase (equal to 1 unit)

g)      1 µL of template DNA (10-100 ng/µL)

 

Enter the following PCR program into a thermal cycler:

 

Temperature (oC)

Time

Cycles

Initial Denaturation

95

3 minutes

1

Denaturation

95

30 seconds

40

Annealing

55

30 seconds

40

Extension

72

45 seconds

40

Final extension

72

10 minutes

1

Hold

4

1

 

 

 

 

 

 

 

 

 

 The NDM gene, if present, will result in a product of 466 base pairs. 

 

Date Created11/08/2013 01:42 PM
Date Updated11/08/2013 01:51 PM

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