1. Quantitative estimation of picoplankton populations in seawater using real time PCR Fei Zhu Daniel Vaulot Station Biologique de Roscoff

2. PCR reaction monitored by SYBR-Green fluorescence 10 ng 1 ng 0,1 ng No template Threshold Quantitative PCR: Principle

3. Standard curve Slope should be close to 3.32 (100% efficiency) Intercept as small as possible (higher sensitivity Ostreococcus with specific primers

4. Summary of previous work (Bastien Simonnet, Masters student) n Primer design –Primer forward unique –Primer reverse will depend on target n Test primer specificity – Problem with Euk primer set initially chosen n Test primer efficiency –Problem with some primer sets n Test on natural sample –encouraging

5. Roscoff sample < 3  m Number of cycles Eukaryotes Mamiellales Micromonas OstreococcusBathycoccus

6. Current work (Fei Zhu post doc - december 2002) n Concentrate on Prasino primers n Test primer specificity n Choose suitable reference n Optimize –extension time –primer concentration –MgCl2

7. Primer set

8. Primer specificity Genomic DNA, regular PCR

9. Standards for Quantitative PCR 18s rRNA PCR Fragment Vector plasmid (pUC18) ccc-Plasmid, Containing 18S rRNA ds- plasmid, containing 18S rRNA Work in terms of gene copies after quantification of DNA concentration with SYBR Green by fluorometry

10. Use of different templates Bathycoccus prasinos EUK528f/Bathy03r

11. Annealing-extension time for different amplicon sizes Bathycoccus prasinos

13. Optimize the concentration of MgCl 2 ds-plasmid from Bathycoccus prasinos EUK528f/Bathy03r

14. What is next ? n Test the specificity of the primers with Q-PCR n Optimize for all primers –MgCl2 concentration –primer concentration n Culture mixes n Environmental samples – Test with internal standards – Process PICODIV samples