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Zebrafish EST Collection

Zebrafish EST Collections

Zebrafish are an important model organism for the study of vertebrate development and disease, organ function, behaviour, and toxicology. Source BioScience is a distributor for the zebrafish myoblast cDNA library (ZF_mu) constructed by Sarah Baxendale (Ingham Lab, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, UK).

To quickly overview the abundance of molecules or potential genes in the library please access the Zebrafish EST search page at the Wellcome Trust Sanger Institute.

Source BioScience offers the complete clone set of 299 384-well microtitre plates or individual clones, for research purposes only. We can also make subsets of your chosen clones from the collection. Please contact us to discuss your requirements.

Protocols / Clone Handling

Plates contain LB broth with 8% glycerol and ampicillin. The plates are sent on Dry Ice, and should be stored at -70°C.

The clones have been streaked onto LB agar containing 50 mg/ml Ampicillin. Please store them at 4°C (not in a freezer).

As soon as possible, re-streak the clones onto the same medium and incubate overnight at 37°C to obtain single colonies.

A selection of at least 10 single colonies should be picked and grown in LB broth containing Ampicillin + 8% glycerol for subsequent freezing at –70°C. These frozen stocks can then be re-grown for plasmid isolation and purification.

Streaking out for single colonies is essential in case of contamination or cross-contamination, or if modifications of the original clone have taken place as the library has been replicated. If there is a problem, the more single colonies you streak out, the more likely it is that you can retrieve the original construct. You can also isolate the original clone from a contaminated sample by following this procedure.

Recommended methods for isolation and purification of DNA

Plasmid DNA mini-prep preparation            

Any standard mini-prep method is suitable. Below is the method used in-house at Source BioScience.

  1. Streak out plasmid onto LB + ampicillin (or chloramphenicol [CM]) plate. Incubate overnight at 37°C (amp.) or 30°C (CM).
  2. Dispense 5 ml LB medium into a 50 ml sterile tube. Add appropriate antibiotic. Inoculate with a single colony and incubate overnight in shaking incubator 37°C [30°C (CM)]/170 rpm.
  3. If a glycerol stock is required, transfer aliquot of 140µl to a 1.5 ml microtube. Add 40µl 80% glycerol. Mix and freeze.
  4. Take 2 ml of the plasmid broth, transfer to a 2 ml microtube, and centrifuge 15 min 3000 rpm.
  5. Pour off supernatant and discard. Re-suspend pellet in 200µl GTE.

Add 5µl RNAase to a stock (10 mg/ml). Incubate 10 min at room temperature.

  1. Lysis: Add 400µl 2M NaOH/1% SDS (freshly made). Mix by inversion. Place on ice for 5 min.
  2. Add 300µl 3M K Ac. Invert to mix. Place on ice for 10 min.
  3. Centrifuge 10 min at 13000 rpm in microfuge. Decant supernatant into a 1.5 ml microtube.

Containing 1 ml cold ethanol, vortex briefly and leave to stand for 30 min on ice.

  1. Centrifuge 10 min at 13000 rpm in microfuge. Remove ethanol carefully (preferably by suction technique).
  2. Add 200µl cold 70% ethanol, vortex briefly, centrifuge 10 min. at 13000 rpm in microfuge, remove ethanol carefully (as above).
  3. Air dry pellet. Add 50µl TE and leave to re-suspend overnight at 4°C.

Solutions      

10 ml GTE: 0.5 ml  20% glucose                           
0.25 ml  1M Tris/HCl pH 7.5
0.2 ml 0.5M EDTA pH 8.0.
9.05 ml sterile distilled water

Lysis Buffer  

100 ml 0.2M NaOH/1% SDS = 0.8 g NaOH
5 ml 20% SDS
95 ml sterile distilled water.

PEG purification of plasmid DNA (strongly recommended before sequencing)

  1. Add an equal volume of 20% PEG solution in 2.5 M NaCl. Incubate at room temperature for 5-10 min. Spin for 10-15 min. at 13,000 rpm.
  2. Remove supernatant and rinse pellet with 70% ethanol. Dry the pellet. Re-suspend in 20 ml sterile distilled water.
  3. Remove 5 ml and add to 2 ml loading buffer. Run out with appropriate DNA marker on a 1.5% TBE agarose gel to check that DNA recovery is good.
  4. The DNA is now ready for sequencing.

General PCR Purification Method

Clones can be amplified by PCR using the appropriate flanking primers. PCR products should then be purified using spin columns or the sAP/exo1 (shrimp alkaline phosphatase/exonuclease1)  method prior to sequencing.

Werle. E, Schneider. C, Renner. M, Volker.M and Fiehn. W. (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucl. Acids Res. 22: 4354-4355.

Hanke. M and Wink. M. (1994) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. BioTechniques 17: 858-860.

References

Although not published, please refer to the following authors and institute:
Title: Sanger Danio rerio EST project
Authors: Humphray,S., Plumb,B., Taylor,R., Francis,M., McLaren,S., Chen,C.-K., Gilbert,J., Rogers,J.
Institute: Ingham Lab, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, UK
Year: 2006

Please acknowledge Sarah Baxendale, MRC Intercellular Signaling Group, University of Sheffield, and Source BioScience LifeSciences in any resulting publications.


For further information and prices please contact us or call +44 (0)115 973 9012

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