PCR detection methods supplement
2013
Supplementary Table 2. Composition of PCR reactions.
Master Mix for the real-time PCR FatA(A) Assay, 126 and 129 bp amplicons. Primer and probe
sequences are shown in Table 1.
Chemicals
Concentration
Final
Concentration*
µl/rxn
TaqMan® Universal PCR
Master Mix, No AmpErase®
UNG
2x
1x
10.0
09-0-3249 (forward primer) 10 µM
300 nM
0.6
09-0-3251 (reverse primer)
10 µM
900 nM
1.8
09-QP-87 (probe)
10 µM
150 nM
0.3
Sterile water
2.3
Total volume
15.0
* Total PCR reaction is 20 µl (15 µl master mix and 5 µl genomic DNA template)
Master Mix for the DP-Ø73496-4 real-time PCR Assay, 84 bp amplicon. Primer and probe
sequences are shown in Table 2.
Chemicals
Concentration
Final
Concentration*
µl/rxn
TaqMan® Universal PCR
Master Mix, No AmpErase®
UNG
2x
1x
10.0
09-0-2824 (forward primer) 10 µM
600 nM
1.2
09-0-2825 (reverse primer)
10 µM
600 nM
1.2
09-QP-83 (probe)
10 µM
250 nM
0.5
Sterile water
2.1
Total volume
15.0
* Total PCR reaction is 20 µl (15 µl master mix and 5 µl genomic DNA template)
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PCR detection methods supplement
2013
Master Mix for the gel-based FatA(A) PCR Assay, 135 and 138 bp amplicons. Primer and probe
sequences are shown in Table 1.
Chemicals
Concentration
Promega 2x PCR Buffer
10 x
Forward primer (11-O4046)
Final
Concentration*
µl/rxn
1x
12.5
10 µM
200 nM
0.5
Reverse primer (11-O-4253) 10 µM
200 nM
0.5
Molecular grade water
9.0
Total volume
22.50
* Total PCR reaction is 25 µl (22.50 µl master mix and 2.5 µl genomic DNA template)
Master Mix for the gel-based DP-Ø73496 PCR Assay (142bp amplicon). Primer sequences are
shown in Table 2.
Final
Concentration*
Chemicals
Concentration
10x PCR Buffer II
10 x
1x
2.5
MgCl2
25 mM
4 mM
4
dNTPs
10 mM
200 µM
0.5
Forward primer (10-O3514)
10 µM
400 nM
1
Reverse primer (10-O-3515) 10 µM
400 nM
1
AmpliTaq Gold® DNA
polymerase
0.625 U/Rxn
0.125
5 U/µL
µl/rxn
Molecular grade water
13.375
Total volume
22.50
* Total PCR reaction is 25 µl (22.50 µl master mix and 2.5 µl genomic DNA template)
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