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Working summary

Working summary. Wang Qian. Analyse the 16SrDNA fragment by DGGE. The samples come from China. DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM , mouse tail protocol) The primers are 16S-SR and 16S-SF-GC. The machine of Dgge is INGENYphorU-2 system.

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Working summary

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  1. Working summary Wang Qian

  2. Analyse the 16SrDNA fragment by DGGE • The samples come from China. • DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol) • The primers are 16S-SR and 16S-SF-GC. • The machine of Dgge is INGENYphorU-2 system.

  3. 2.Work for Artemia • DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol) • The samples are listed( table 1)

  4. Table 1

  5. Optimize of pcr condition • The primers are 12S-SP and 12S-SF-GC. • Choose appropriate melting temperature: The Tm of 12S-sp is 43.03℃ The Tm of 12S-sfgc is 77℃ • The anneal temperature of gradient are 45℃, 48℃, 51℃ and 55℃(Fig.1). • The components of pcr (table. 2). • The thermal cycle condition is list (table.3)

  6. Table.2 (Jump start mix Sigma kit)

  7. Table.3

  8. The ampilfication of DNA • The number of ARC: 1039,1366,1377,1378,1387,1389,1356, 1016,1367,1163,1162,1380,1371,1405, 1406(fig.2.) • Salinas Grandes Cordova population 1-15(fig.3). • Lasana Cisne population 16-30(fig.3).

  9. fig. 2

  10. Fig.3

  11. The components of pcr(the kit of Taq DNA Polymerase)

  12. Fig.4

  13. Analysis of pcr products by DGGE • Gel: 9% • Den. Gradient: 15%-40% • Runing: 16h at 57 ℃, at 120v • Stain: SYBR golden

  14. Analysis of pcr products by DGGE • Gel: 9% • Den. Gradient: 20%-60% • Runing: 16h at 57 ℃, at 120v • Stain: SYBR golden

  15. Check concentration of Dna • concentration of Dna was diluted 50ng/ul • Dilution Dna 1,10,100,1000,10000 times • PCR • Check concentration of Dna • Loading the samples 50ng for DGGE.

  16. Result: • DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol) • check concentration of DNA • DNA was diluted at 50ng/ul • dilution DNA 1000 times • Purified the productions of PCR

  17. Amplification (the kit of Taq DNA Polymerase)

  18. 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 94℃, 30s; 51℃,30s; 72℃,1m, 26 cycle or 28 cycle 72℃, 4m, 1 cycle

  19. DGGE (1) Gel%: 9% (2) Den. gradient: 15%-45% (3) Voltage: 120v (4) Runtime: overnight; about 16h (5) Staining: SYBR gold (6) loading sample: 50ng production of PCR

  20. Project of working in the next phage • To try make use of Purified the production of PCR to do DGGE • To find appropriate the ladder of Artemia • To do more samples , at the same time, adjust the protocols of PCR and DGGE.

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