Working summary Wang Qian Analyse the 16 Sr

Working summary Wang Qian

Analyse the 16 Sr. DNA 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 16 S-SR and 16 S-SF-GC. • The machine of Dgge is INGENYphor. U-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 number The name of Artemia 1218 A. sinica 1226 A. urmiana 1258 A. franciscana 1268 A. salina 1321 A. persinmilis

Optimize of pcr condition • The primers are 12 S-SP and 12 S-SF-GC. • Choose appropriate melting temperature: The Tm of 12 S-sp is 43. 03℃ The Tm of 12 S-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(p. H 8. 3) 10 m. M Mg 2+ 2 m. M d. NTP 0. 2 m. M primers Each 1 u. M Taq 0. 6 u DNA 1 ul Total 20 ul

Table. 3 Step 1 94 ℃ 2 m Step 2 94 ℃ 30 s 45 ℃; 48 ℃; 51 ℃; 55 ℃ 30 s Step 3 72 ℃ 1 m 72 ℃ 2 m 1 cycle 30 cycle 1 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 115(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(p. H 8. 3) 10 m. M Mg 2+ 2. 5 m. M d. NTP 0. 2 m. M primers Each 0. 25 u. M BAS 1 x Taq 0. 2 u DNA 1 ul Total 20 ul

Fig. 4

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

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

• • • Check concentration of Dna was diluted 50 ng/ul Dilution Dna 1, 100, 10000 times PCR Check concentration of Dna Loading the samples 50 ng 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 50 ng/ul • dilution DNA 1000 times • Purified the productions of PCR

• Amplification (the kit of Taq DNA Polymerase) item Tris-HCl(p. H 8. 3) Mg 2+ d. NTP primers BAS Taq DNA Total Final concentration 10 m. M 2. 5 m. M 0. 2 m. M Each 0. 25 u. M 1 x 0. 2 u Dilution 1000 times DNA, 4 ul 40 ul

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

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