work summary

Wangqian

2007-01-12

Mix samples(ARC1258 and ARC1218)

0/100 1/99 5/95 10/90 50/50 90/10 95/5 99/1 100/0

1258A.franciscana

0mg 0.6mg 2.6mg 5mg 25mg 45mg 47.4mg 49.5mg 50mg

1218A.sinica

50mg 49.4mg

47.6mg 45.2mg 25mg 5mg 2.5mg 6mg 0mg

total 50mg 50mg 50.2mg 50.2mg 50mg 50mg 49.9mg 50.1mg 50mg

• DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol)

• DNA dilute 20 times to PCR, then DNA concentration is unified to 20ng/ul.

• 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℃ 26x, 28x, 30x94 , 30s; 51 ,30s; 72 ,1m, ℃ ℃ ℃ 72 , 4m , 1 cycle ℃

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, 5ul

Total 50ul

PCR (0/100 and 100/0)

• Dilute DNA(20ng/ul) 0 time, 10times, 100times, 1000times.

• Amplication 30cycle, 28cycle, 26cycle.

Count the cyst with magnifier

0/100 1/99 5/95 10/90 50/50 90/10 95/5 99/1 100/0

1258A.franciscana

0 1 5 10 50 90 95 99 100

1218A.sinica

100 99 95 90 50 10 5 1 0

total 100 100 100 100 100 100 100 100 100

• DNA extraction : use the Wizard Genomic DNA Purification kit (Promega TM, mouse tail protocol)

• DNA concentration is 10ng/ul.

• Count cyst again, extract the DNA with Chelex 6%(Bio-Rod).

• 0/100 and 100/0

• Use 3ul of supernatant for 50ul PCR reaction.

• 26cycle,28cycle,30cycle,32cycle,34cycle,36cycle.

DNA extracted by chelex 6%

Promega and chelex 6%

• use samples from ghent and dvz

• Mix by 50/50

• Extract DNA by promage, concentration of DNA are 9.5ng/ul and 21ng/ul. then dilute DNA to 9.5ng/ul.

• Dilute DNA 1000times to PCR

• 32cycle