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| Project C 4 |
| Part 4: DNA extraction from infested beech roots |
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All PCR-inhibiting plant metabolites must be removed and DNA extraction has to be quantitative and reproducible. We tested several DNA extraction protocols with Phytophthora infested beech roots and showed that reproducible results were obtained, when the extracted DNA was finally cleaned up with wizard DNA clean up kit from Promega or with a PVPP-spin column (see table 2).
Table 2:
DNA extraction protocols (+ suitable, - not suitable for quantitative PCR)
Table 3 shows that the DNA-extraction of P. citricola infected beech roots with the DNeasy kit from Qiagen in combination with the wizard DNA clean up kit gave highly reproducible results. The Ct-values of four independent DNA-extractions of the same root powder were almost identical.
Table 3:
Ct-values after quantitative SYBR-green PCR od P. citricola infested 2 year old beech roots
The recovery rate for DNA from mycelia using the wizard DNA clean up kit was 100 % when Ct-values were compared (table 4).
Table 4:
Ct-values after TaqMan PCR of P. citricola mycelia with and without the wizard DNA clean up kit. (n=3)
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