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Dye terminator cycle sequencing (DTCS) reactions incorporate many components including DNA polymerase, pyrophosphatase, buffer, dNTPs, dye terminators, DNA templates and primers. The stepwise addition of so many components is time consuming and if done manually, can introduce errors associated with pipetting small volumes and viscous solutions.
The GenomeLab DTCS Quick Start Kit simplifies this process by using a master-mix of many of the components -- reducing the number of pipetting steps from 10 to 4 while using larger transfer volumes to reduce pipetting error. Each kit provides enough reagents to complete 100 sequencing reactions.
CEQ performance using the GenomeLab DTCS Quick Start Kit is comparable to that achieved with the GenomeLab Methods Development Kit using the LFR-1 method. Table 1 represents an average of 24 individual samples run using this chemistry and summarizes the percent accuracy calculated at 500, 550, 600 and 700 bases. Accuracy is calculated using a call threshold setting of zero, which disallows all ambiguous calls (N), providing a more accurate yet conservative estimate of read length. Table 2 represents the same analysis using 1/2 X and 1/4 X reactions*.
Table 1
| Sample |
# of Bases |
Accuracy |
| pUC18 50 fmol |
500 |
99.12% |
| |
550 |
99.18% |
| 600 |
99.27% |
| 700 |
99.24% |
| pUC18 100 fmol |
500 |
99.30% |
| |
550 |
99.36% |
| 600 |
99.41% |
| 700 |
99.33% |
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Table 2
| Sample |
# of Bases |
Accuracy |
| pUC18 1/2 reaction |
500 |
99.49% |
| |
550 |
99.53% |
| 600 |
99.56% |
| 700 |
99.38% |
| pUC18 1/4 reaction |
500 |
99.34% |
| |
550 |
99.22% |
| 600 |
99.05% |
| 700 |
98.61% |
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* Beckman Coulter recommends using full reaction concentrations to achieve the best system performance over a wide array of sample templates.
Specifications
 GenomeLab DTCS Quick Start Kit
Contains volumes required for 100 reactions: |
Quick Start Mix
- dATP, dCTP, dTTP, dITP
- ddUTP, ddGTP, ddCTP, ddATP (WellRED label)
- Tris –HCL, MgCl2 , reaction buffer - pH 8.9
- Thermo Sequenase DNA Polymerase*
- Pyrophosphatase |
800 µl |
| (-) 47 Sequencing Primer |
240 µl |
| pUC18 Control Template |
20 µl |
| Glycogen |
110 µl |
| Mineral Oil |
5 ml |
| Sample Loading Solution (SLS) |
6 ml |
| ** Although equivalent readlength/accuracy performance have been demonstrated between the standard GenomeLab Methods Development Kit and the GenomeLab DTCS Quick Start Kit, slight variations in the electropherogram may exist, as they utilize different DNA polymerases. |
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