Human cystic fibrosis clone collection


Please note that the clone(s) being sent have tested negative in our T1 phage contamination assay. However these clones should still be handled with care as no phage assay can be guaranteed to be 100% reliable.

THE DISPATCH NOTE SHOWS THE ORIGINAL CLONE ID ORDERED AND ALSO THE ALIAS (PLATE AND WELL NUMBER ). THIS IS WHAT IS SHOWN ON THE CLONE LABEL. PLEASE KEEP THE DISPATCH NOTE FOR FUTURE REFERENCE.
WHAT TO DO WITH YOUR CLONES WHEN YOU RECEIVE THEM


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

Individual clones have been streaked onto LB agar containing 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 appropriate antibiotic + 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.

Tissue source- All tissues were obtained from Dr. M. J. Welsh, University of Iowa College of Medicine. All libraries were created by Bento Soares for the program University of Iowa and Cystic Fibrosis Foundation - Cystic Fibrosis Project.

Library construction-Libraries were constructed and/or subtracted according to Bonaldo, Lennon and Soares, Genome Research, 6:791-806, 1996. First strand cDNA synthesis was primed with an oligo-dT primer containing a Not I site. Double stranded cDNA was ligated to an EcoR I adaptor, digested with Not I, and cloned directionally into pT7T3-Pac vector. The oligonucleotide used to prime the synthesis of first-strand cDNA contains a library tag sequence that is located between the Not I site and the (dT)18 tail.

 

Primary human lung epithelial cells, various treatments


Tissue Description : A pool of 10 adult human lung epithelial cell samples; day 0 suspension,
untreated control, heat shock treated, 95% oxygen treated, dexamethasone and T3 treated,
heregulin treated, LPS treated, serum free media treated, KGF treated, and low temperature
treated.

Normal human lung tissue, pool of different developmental stages

Tissue description : A pool of 7 human lung cell samples from different developmental
stages; fetal day 64 lung tissue, fetal day 87 lung tissue, fetal week 19 lung tissue, adult 30
year old normal distal lung tissue, adult 78 year old normal alveoli lung tissue, fetal week
42 lung tissue, fetal week 42 lung tissue.

Start Library Name: EC0
Normalized Library Name: EC1
Library Tag: AAGTGCTTAC
Lab host: DH10B (Life Technologies) (T1 phage resistant)
Vector: pT7T3-Pac (Pharmacia) with a modified polylinker
R. Site 1: EcoR I
R. Site 2: Not I

 

Primary human lung cystic fibrosis epithelial cells, various treatments

Tissue description : A pool of adult human cystic fibrosis lung epithelial cells from 3 specimens,
each specimen untreated, treated with LPS for 6 hours, and treated with LPS for 24 hours.


Start Library Name: EN0
Normalized Library Name: EN1
Library Tag: CTGCTCAGGT
Lab host: DH10B (Life Technologies) (T1 phage resistant)
Vector: pT7T3-Pac (Pharmacia) with a modified polylinker
R. Site 1: EcoR I
R. Site 2: Not I

 

Human lung epithelial cells (pooled and subtracted library #1)

FN0 is a subtracted library derived from a pool of two normalized human lung
libraries: normalized Primary Human Lung Epithelial Cells, Various Treatments (DU1) and
normalized Primary Human Lung Cystic Fibrosis Epithelial Cells, Various Treatments (EN1).

Subtracted Library Name: FN0
Library Tags: GGCTGTAGGC, CTGCTCAGGT
Lab host: DH10B (Life Technologies) (T1 phage resistant)
Vector: pT7T3-Pac (Pharmacia) with a modified polylinker
R. Site 1: EcoR I
R. Site 2: Not I


Reference

Bonaldo, Lennon and Soares, Genome Research, 6:791-806, 1996

 

Acknowledgements

We are grateful to Dr. M. Bento Soares and colleagues (University of Iowa) for the gift of this library. Our thanks are also due to the following members of University of Iowa:

 

Dr. M. J. Welsh (Tissue Procurement), Dr. M. Bento Soares (cDNA Library preparation, library arraying and sequencing), Dr. P. B. McCray (EST data submission) and Tamara A. Kucaba for replication of the library.