Tissues:
The following tissues were removed from xx freshly killed adult Atlantic halibut (both males and females) reared at St. Andrews Biological Station:
- Gill
- Liver
- Skin
- Ovary
- Intestine
- Testis
- Head kidney
- Spleen
The tissues were placed in RNALater (Ambion) and placed at -20 °C overnight. Tissues were then frozen at -80 °C until used.
mRNA was isolated from 1g of pooled tissue using the Fast Track 2.0 mRNA Isolation Kit (Invitrogen ).
Larval samples:
Larvae were sampled at Scotian Halibut Ltd. and placed in RNALater at -20 °C overnight. The following stages were used:
- Hatch
- Mouth-opening
- Midway to metamorphosis
- Pre-metamorphosis
- Post-metamorphosis
Total RNA was isolated from 5-20 individual larvae (depending on the stage) using Trizol.
cDNA synthesis:
cDNA was synthesized from either mRNA (0.25-0.5 µg) or total RNA (1-2 µg) using the SMART protocol (Clontech)
Normalization:
cDNAs were normalized using the Trimmer Direct kit (Evrogen)
Cloning:
Normalized cDNAs were digested with SfiI, size fractionated to remove products under 500 bp, and directionally cloned into the SfiI site of pDNR-Lib (Clontech).
Transformation:
Ligated DNA was transformed into ElectroMAX DH10B T1 Phage-resistant E. coli by electroporation and plated on LB plates containing 30 µg/ml chloramphenicol. 96 colonies were randomly screened by PCR for insert size and recombination efficiency.
Libraries with greater than 95% recombination efficiency were amplified using the semi-solid amplification method (refer to Stratagene's "pBLUESCRIPT II XR cDNA Library Construction Kit Instruction Manual - pg 27-28; Gibco BRL's "Guide to Bacterial Transformations with Chemically and Electrotransformable Competent Cells" - pg 20) and stored at -80 °C in 2X LB/12.5% glycerol.
