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PCR |
| Name: |
Cas_PAL |
Type: |
Real-Time PCR |
System: |
Quantitative |
| Genetic Element: |
phenylalanine ammonia-lyase (PAL) |
Ampl. Length: |
144 |
Ampl. Position: |
|
Sequence Size [bp]: |
|
| AnnTemp [°C]: |
60 |
Taxonomy: |
Chestnut: Sweet Chestnut: Castanea vesca |
Temp. Time Program: |
Process Time [min:s] Temperature [°C]<br>Pre-PCR: decontamination 02:00 50<br>Pre-PCR: activation of DNA polymerase and denaturation of template DNA 10:00 95<br>PCR (45 cycles) <br>Step 1 Denaturation 00:15 95<br>Step 2 Annealing and elongation 01:00 60 |
| Device: |
|
Reagents: |
Total volume 10 µl<br>Template DNA (maximal amount 200 ng) 2 µl<br>DNA polymerase AmpliTaq Gold® 1,5 IU<br>Decontamination system dUTP<br>AmpErase uracil N-glycosylase 400 µmol/l<br>0,5 IU<br>Reaction buffer TaqMan® buffer A (containing passive reference ROX)a 1 X<br> MgCl2 7<br>Primers 300 900<br>dNTP dATP, dCTP, dGTP 200 µmol/l each200 200 nmol/l<br> |
nPrimers: |
3 |
Organisation: |
BfR |
| PCR Description: |
|
Sequence: |
|
| LOD [copy]: |
10 |
LOQ [copy]: |
100 |
Efficiency [%]: |
90 |
| Remarks: |
|
Primers |
| PPName: |
Type: |
PrimerSequence: |
Length [bp]: |
T_Ann [°C]: |
TmThermo [°C]: |
Tm2AT [°C]: |
GC [%]: |
Position: |
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Cas_PAL-f |
Sense |
CCA AAg AAg TAg Agg gTg CAA gA |
23 |
67.2 (PCR: 60) |
58.4 |
68 |
47.83 |
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Cas_PAL-r |
Antisense |
gAA TTC TCT CAC CAg TTA gTA AAC TTg TT |
29 |
65.5 (PCR: 60) |
56.7 |
78 |
34.48 |
|
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Cas_PAL-probe |
Marker/Probe |
(FA M)A AAG CAG CAA TTC CTA ACC CAA TTA AGG AGT (TA MRA ) |
31 |
72.1 (PCR: 60) |
65.4 |
86 |
38 |
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