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 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 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 4

5 , 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

Mg2+ 2mM

dNTP 0.2mM

primers Each 1uM

Taq 0.6u

DNA 1ul

Total 20ul

Table.3

Step 1 94 ℃ 2m 1 cycle

Step 2 94 ℃ 30s

45 ; 48 ; 51 ; 55 ℃ ℃ ℃℃

30s 30 cycle

72 ℃ 1m

Step 3 72 ℃ 2m 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 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

Mg2+ 2.5mM

dNTP 0.2mM

primers Each 0.25uM

BAS 1x

Taq 0.2u

DNA 1ul

Total 20ul

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

Mg2+ 2.5mM

dNTP 0.2mM

primers Each 0.25uM

BAS 1x

Taq 0.2u

DNA Dilution 1000 times DNA, 4ul

Total 40ul

• 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.