Embed Size (px)
Citation preview
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.
