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| <p><strong>Normalized Cheyenne wheat unstressed endosperm cDNA library (Deshui Zhang, Henry T. Nguyen's Lab, TTU, March 1, 2001)</strong></p> |
| <p><strong>Source of non-normalized cDNA library</strong></p> |
| <p>- Cheyenne wheat unstressed seedling shoot cDNA library, from Dr. Anderson's Lab</p> |
| <p>- Mass excised, amplified phagemid library</p> |
| <p>- Titer of the phagemid library: 3 x 108 cfu/ml on TJC 121 cells (from Dr. Close).</p> |
| <p><strong>Description of normalized cDNA library</strong></p> |
| <p><em>Procedures</em></p> |
| <p>- Amplification of the phagemid library with TJC121 cells;</p> |
| <p>- Isolation of single-stranded phagemid DNA through the infection of VCSM13 helper phage (Stratagene Company);;</p> |
| <p>- Pvu II restriction digestion of single-stranded phagemid DNA;</p> |
| <p>- Purification of Pvu II digested single-stranded phagemid DNA using hydroxylapatite (HAP) chromatography;</p> |
| <p>- Preparation of driver cDNA through PCR amplification of purified single-stranded phagemid DNA;</p> |
| <p>- Reassociation hybridization between purified single-stranded phagemid DNA and driver cDNA;</p> |
| <p>- Purification of non-hybridized single-stranded phagemimd DNA (normalized) through HAP column;</p> |
| <p>- Conversion of the normalized and single-stranded phagemid DNA into partial duplex form of DNA;</p> |
| <p>- Electroporation transformation</p> |
| <p><em>Host E. coli strain</em>: ElectroMAX DH1OB (BRL Company)</p> |
| <p><em>Titers of normalized cDNA library</em></p> |
| <p>Primary library titer: 1.2 x 105 cfu/ml;</p> |
| <p>Amplified library titer: 3.5 x 107 cfu/ml</p> |
| <p><em>Storage</em>: LB broth with 20% of glycerol and 75 mg/ml of ampicillin at -80 C</p> |
| <p>EMBED Photoshop.Image.5 \s </p> |
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