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Materials and Methods

PCR

Primers fw1 and rev1 are projected on-line in a data base; i aligned  vasa-like genes sequences known of various organism.

FW1 : ATG GCN TGY GCN CAR ACN G

REV1 :RAA NCC CAT RTC NAG CAT

Reaction mix:

0.25 m l cDNA

     1 m l buffer 10x

 0.3 m l MgCl2 50 mM

    1 m l fw1 10 mM

    1 m l rev1 10 mM

0.2 m l dNTP 200 mM

0.25m l Taq Dynazime 0.25 units

 5.7 m l H2O

______

10 m l tot

After Dynazime Taq was actived at 95°C for 5 min , 30 cycles were conducted, with each cycle consisting of 30 sec at 94°C, 1 min at 50 °C and 1 min at 72°C except that the last elongation  reaction was carry out for 8 min.

Primer fw2 was projected on bases of the first fragment sequence obtain from the previous PCR; rev2 was projcted on-line aligning vasa-like genes sequences known

FW2 : GAG GCT GAC AGG ATG TTG GAC ATG GG

REV2 : CCD ATN CGR TGN ACR TAY TC

Reaction mix:

0.25 m l cDNA

     1 m l buffer 10x

 0.3 m l MgCl2 50 mM

    1 m l fw2 10 mM

    1 m l rev2 10 mM

0.2 m l dNTP 200 mM

0.25m l Taq Dynazime 0.25 units

 5.7 m l H2O

______

10 m l tot

After Dynazime Taq was actived at 95°C for 5 min , 30 cycles were conducted, with each cycle consisting of 30 sec at 94°C, 1 min at 50 °C and 1 min at 72°C except that the last elongation  reaction was carry out for 8 min.

 

NORTHEN BLOT

I run about 20 mg/ml of total RNA extract from gonad, liver, muscle in a denaturing gel formaldehyde 0.41 M; for positive control i usued a plasmid (pGEM T easy) with insert the first fragment obtain by PCR. RNAs were blotting in nytrocellulose filter and the filter hibridyzed overnight with a probe label with DIG .