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Chicken EST Collection

Chicken EST Collection

This project to produce a large collection of EST clones from the chicken was funded by the BBSRC and was directed by colleagues at the Universities of Nottingham, Dundee and UMIST. Sequencing was by Incyte Genomics Inc. All tissues were taken from the White Leghorn (Layer) breed (Gallus domesticus). The Adult Cerebrum, Adult Cerebellum and Adult Brain Other Parts tissues were from the White Leghorn IAH strain of RPL Line 15I.
All embryonic tissues were from the White Leghorn 'Hisex' breed. The remaining adult tissues (except muscle, chondrocyte, ovary, pancreas and adipose) originate from White Leghorn Line 15 hens.
Note: IAH = Institute of Animal Health, RPL = Regional Poultry Laboratory, East Lansing, Michigan. For more information please see the Chicken EST Repository Homepage (http://www.chick.umist.ac.uk/).

21 tissues have been sequenced, with each tissue having multiple cDNA libraries produced from different normalisation stringencies. The majority of the clones are in pBluescript II KS+ (Stratagene) in DH10B or DH10B-T1 resistant bacteria. EST sequences have been produced from the 5' end of each clone.

Clones are currently supplied individually, with no limits on the numbers that may be ordered. Clones can also be supplied in our sequence verified and purified plasmid formats. For supply of clones in other formats (e.g. whole plates) please contact us.

Protocols / Clone Handling

The clones have been streaked onto LB agar containing 50 µg/ml carbenicillin. 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 carbenicillin + 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 LifeSciences.

  1. Streak out plasmid onto LB + ampicillin Incubate overnight at 37°C
  2. Dispense 5ml LB medium into a 50ml sterile tube. Add appropriate antibiotic. Inoculate with a single colony and incubate overnight in shaking incubator 37ºC /170 rpm.
  3. If a glycerol stock is required, transfer aliquot of 140µl to a 1.5ml microtube. Add 40µl 80 % glycerol. Mix and freeze.
  4. Take 2ml of the plasmid broth, transfer to a 2ml microtube, and centrifuge 15 min 3000 rpm.
  5. Pour off supernatant and discard. Re-suspend pellet in 200µl GTE.
    Add 5µl RNAase A stock (10mg/ml). Incubate 10 min. room temperature.
  6. Lysis: Add 400µl 0.2M NaOH/1% SDS (freshly made). Mix by inversion. Place on ice for 5 min.
  7. Add 300µl 3M K Ac. Invert to mix. Place on ice for 10 min
  8. Centrifuge 10 min at 13000 rpm in microfuge. Decant supernatant into a 1.5ml microtube
    containing 1ml cold ethanol, vortex briefly and leave to stand for 30 min. on ice.
  9. Centrifuge 10 min. at 13000 rpm in microfuge. Remove ethanol carefully (preferably by suction technique).
  10. Add 200µl cold 70% ethanol, vortex briefly, centrifuge 10 min. at 13000 rpm in microfuge, remove ethanol carefully (as above).
  11. 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
5ml 20% SDS
95ml sterile distilled water.

PEG purification of plasmid DNA (strongly recommended before sequencing)

  1. Add an equal volume of 20% PEG solution in 2.5M 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. Resuspend in 20µl sterile
    distilled water.
  3. Remove 5µl and add to 2µl 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

Please cite 'Boardman PE, Sanz-Ezquerro J, Overton IM, Burt DW, Bosch E, Fong WT, Tickle C, Brown WRA, Wilson SA and Hubbard SJ. A Comprehensive Collection of Chicken cDNAs. Current Biology 2002 12:1965-1969.' when using the EST data. [Medline] and Hubbard SJ, Grafham DV, Beattie KJ, Overton IM, McLaren SR, Croning MDR, Boardman PE, Bonfield JK, Burnside J, Davies RM, Farrell ER, Francis MD, Griffiths-Jones S, Humphray SJ, Hyland C, Scott CE, Tang H, Taylor RG, Tickle C, Brown WRA, Birney E, Rogers J, and Wilson SA Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags Genome Res. 2005 15: 174-183, [Medline]for cDNA and other info.


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