Abstract - The lift pool method for isolation of cDNA clones from lambda phage libraries

The lift pool method for isolation of cDNA clones from lambda phage libraries

Janine LeBlanc-Straceski*
Department of Biology and Allied Health
Merrimack College
North Andover Massachusetts 01845
Te: 978 837 5000 / 4357
Fax: 978 837 5180
E-mail: Janine.LeBlancStraceski@merrimack.edu

Pablo Sobrado
University of Wisconsin, Madison
Biochemistry Department
433 Babcock Drive Room 123
Madison, WI 53706
Tel: 608 262 3364 Ext 2110
E-mail: psobrado@biochem.wisc.edu

Sharon Betz
Clinical Molecular Genetics Fellow
McLendon Clinical Labs
Department of Pathology and Laboratory Medicine
University of North Carolina CB #7525
Chapel Hill, NC 27599
Tel: 919 966 4408
Fax: 919 966 6351
E-mail: sbetz@unch.unc.edu

Julie Zerfas
Building K.
Wyeth BioPharma
1 Burtt Rd., Andover, MA 01810
Tel: 978 247 4295
Fax: 978 2474298
E-mail: jzerfas@wyeth.com

Karen Morgan
College of Osteopathic Medicine
University of New England
11 Hills Beach Road
Biddeford, ME 04005-9599
E-mail: kmorgan1@pipeline.une.edu

*Corresponding author

Financial support: The Faculty Development Grant Program of Merrimack College, Merck/AAAS Undergraduate Science Research Program grant, a National Science Foundation-Undergraduate Research Institution grant (NSF-URI) NSF0077516.

Keywords: cDNA, lambda, non-radioactive, PCR, plaque, screening.

A PCR based strategy was developed to screen a Xenopus oocyte λgt10 cDNA library. The PCR-based lift pool (LP) method follows the same two tiered strategy as conventional screening of phage libraries by filter hybridization. Two rounds of plating, one at high density to detect the clone, and one at low density to purify the clone to homogeneity, are performed. In the first round, lysates from high density plates, termed plate pools (PP), serve as template for PCR. In the second round, phage particles adhering to plaque lifts of low density plates are washed off nitrocellulose filters to create LPs, which are used as template for PCR. The integrity of the plaques on the low-density plates is preserved. Once a positive LP has been identified, plaques on the corresponding plate are screened individually by PCR. Using isoform specific primer pairs for Xenopus myosin 7a and myosin 1d, two lambda clones were isolated. Subsequent DNA sequence analysis confirmed their identities as myosin isoforms (GenBank accession numbers: DQ100353 and AF540952). This method offers a time saving, cost-effective alternative to other hierarchical pooling strategies for the repeated screening by PCR of an arrayed lambda phage library.