Figure 1 A representative RT-PCR assay plot (A) and standard curve (B) for detection of chloroplast DNA.
A. Amplification plot shows PCR product of four wheat DNA
samples used to generate the standard curve, one marijuana positive
control and two unknown marijuana samples for quantitation.
The negative control did not amplify in the plot as expected.
B. Standard Curve [cycle threshold number (Ct) x DNA concentration
(Log CO)]
Replicate1 |
Replicate2 |
Replicate2 |
||||||
|
Ct |
Qty |
|
Ct |
Qty |
|
Ct |
Qty |
Standard 1 |
22.81 |
13.25ng/pL |
Standard 1 |
22.48 |
13.25ng/pL |
Standard 1 |
21.94 |
13.25nghth |
Standard 2 |
25.18 |
5.3ng/p1 |
Standard 2 |
23.23 |
5.3ng/p1 |
Standard 2 |
23.63 |
5.3ng/pL |
Standard 3 |
26.01 |
2.65ng/p1 |
Standard 3 |
25.09 |
2.65ng/pL |
Standard 3 |
25.05 |
2.65ng/pL |
Standard 4 |
27.47 |
1.99ng/pL |
Standard 4 |
25.72 |
1.99ng/pL |
Standard 4 |
24.79 |
1.99ng/pL |
Positive control |
26.3 |
2.9 ing/pL |
Positive control |
25.72 |
1.77ng/pL |
Positive control |
25.99 |
1.19ng/pt |
Standard Curve |
Standard Curve |
Standard Curve |
||||||
Slope: |
-5.21 |
Slope: |
-4.03 |
Slope: |
-3.77 |
|||
R2 |
0.96 |
R2 |
0.935 |
R2 |
0.95 |
|||
Table 1: Results for three replicate RT-PCR assays using the rbcL PCR primers for detection. Ct values represent the PCR cycle number at which the sample crossed the analytical threshold of the amplification plot and Qty values represent the concentrations of the samples. Replicate 3 meets the quality control criteria for the assay.
|
Sample |
Ct |
Qty |
Replicate3 |
MJ3 (marijuana |
21.5 |
18.43 ng/pl, |
MJ4 (marijuana) |
25.77 |
1.36 ng/pL |
|
|
MJ10 (marijuana) |
25.99 |
1.19 ng/pL |
Table 2: Results of the RT-PCR assay for detection of positive control (MJ10) and evidentiary marijuana samples (MJ3, MJ4). The Ct value represents the cycle number at which the sample crossed the threshold of the amplification plot. The Qty value represents the calculated DNA concentration of each of the samples when plotted against the wheat DNA standard curve.