1. work summary Wangqian 2007-01-12

2. Mix samples(ARC1258 and ARC1218) 0/1001/995/9510/9050/5090/1095/599/1100/0 1258 A.franc iscana 0mg0.6mg2.6mg5mg25mg45mg47.4mg49.5mg50mg 1218 A.sinic a 50mg49.4m g 47.6mg45.2mg25mg5mg2.5mg6mg0mg total 50mg 50.2mg 50mg 49.9mg50.1mg50mg

3. DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol) DNA dilute 20 times to PCR, then DNA concentration is unified to 20ng/ul.

4. The primers are 12S-SP and 12S-SF-GC 12S-SP: 5’-CTA-GGA-TTA-GAT-ACC-CTA-3’ 12S-SF-GC: 5’-CCG-GGG-CCC-GCG-GGC- CCC-CGG-GCC-GGG-CCC-GGG-GAG-AGC- GAC-GGG-CGT-ATG-TAT-3’ PCR condition: 94 ℃, 2m, 1 cycle 26x, 28x, 30x94 ℃, 30s; 51 ℃,30s; 72 ℃,1m, 72 ℃, 4m, 1 cycle

5. Amplification (the kit of Taq DNA Polymerase) item Final concentration Tris-HCl(pH 8.3) 10mM Mg 2+ 2.5mM dNTP0.2mM primersEach 0.25uM BAS1x Taq0.2u DNADilution 1000 times DNA, 5ul Total50ul

6. PCR (0/100 and 100/0) Dilute DNA(20ng/ul) 0 time, 10times, 100times, 1000times. Amplication 30cycle, 28cycle, 26cycle.

9. Count the cyst with magnifier 0/1001/995/9510/9050/5090/1095/599/1100/0 1258 A.franciscana 0151050909599100 1218 A.sinica 1009995905010510 total 100

10. DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol) DNA concentration is 10ng/ul. Count cyst again, extract the DNA with Chelex 6%(Bio-Rod).

12. 0/100 and 100/0 Use 3ul of supernatant for 50ul PCR reaction. 26cycle,28cycle,30cycle,32cycle,34cycle,3 6cycle. DNA extracted by chelex 6%

14. Promega and chelex 6%

17. use samples from ghent and dvz Mix by 50/50 Extract DNA by promage, concentration of DNA are 9.5ng/ul and 21ng/ul. then dilute DNA to 9.5ng/ul. Dilute DNA 1000times to PCR 32cycle