Normalized Cheyenne wheat unstressed endosperm cDNA library (Deshui Zhang, Henry T. Nguyen's Lab, TTU, March 1, 2001)

Source of non-normalized cDNA library

- Cheyenne wheat unstressed seedling shoot cDNA library, from Dr. Anderson's Lab

- Mass excised, amplified phagemid library

- Titer of the phagemid library: 3 x 108 cfu/ml on TJC 121 cells (from Dr. Close).

Description of normalized cDNA library

Procedures

- Amplification of the phagemid library with TJC121 cells;

- Isolation of single-stranded phagemid DNA through the infection of VCSM13 helper phage (Stratagene Company);;

- Pvu II restriction digestion of single-stranded phagemid DNA;

- Purification of Pvu II digested single-stranded phagemid DNA using hydroxylapatite (HAP) chromatography;

- Preparation of driver cDNA through PCR amplification of purified single-stranded phagemid DNA;

- Reassociation hybridization between purified single-stranded phagemid DNA and driver cDNA;

- Purification of non-hybridized single-stranded phagemimd DNA (normalized) through HAP column;

- Conversion of the normalized and single-stranded phagemid DNA into partial duplex form of DNA;

- Electroporation transformation

Host E. coli strain: ElectroMAX DH1OB (BRL Company)

Titers of normalized cDNA library

Primary library titer: 1.2 x 105 cfu/ml;

Amplified library titer: 3.5 x 107 cfu/ml

Storage: LB broth with 20% of glycerol and 75 mg/ml of ampicillin at -80 C

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