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.

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

Table 1 ARC of numberThe name of Artemia 1218A.sinica 1226A.urmiana 1258A.franciscana 1268A.salina 1321A.persinmilis

Optimize of pcr condition The primers are 12S-SP and 12S-SF-GC. Choose appropriate melting temperature: The Tm of 12S-sp is ℃ 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)

Table.2 ( Jump start mix Sigma kit ) item Final concentration Tris-HCl(pH 8.3) 10mM Mg 2+ 2mM dNTP0.2mM primersEach 1uM Taq0.6u DNA1ul Total20ul

Table.3 Step 1 94 ℃ 2m1 cycle Step 2 94 ℃ 30s 45 ℃ ; 48 ℃ ; 51 ℃ ; 55 ℃ 30s30 cycle 72 ℃ 1m Step 3 72 ℃ 2m1 cycle

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).

fig. 2

Fig.3

The components of pcr (the kit of Taq DNA Polymerase) item Final concentration Tris-HCl(pH 8.3) 10mM Mg mM dNTP0.2mM primersEach 0.25uM BAS1x Taq0.2u DNA1ul Total20ul

Fig.4

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

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

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.

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

Amplification (the kit of Taq DNA Polymerase) item Final concentration Tris-HCl(pH 8.3) 10mM Mg mM dNTP0.2mM primersEach 0.25uM BAS1x Taq0.2u DNADilution 1000 times DNA, 4ul Total40ul

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

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

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.