ASH is APA-based, full-length-enriched, single-stranded cDNA subtraction hybridization library.
ASH library specifications
• Insert sizes of about 1200bp (based on colony PCR with 10% of clones <500bp, and a >95% recombinant rate.
• Number of clones: >30,000 for a subtracted cDNA library. Additional clone available upon request for an extra fee.
• Full-length enriched.
• Directional cloning: Subtracted cDNA will be ligated directionally in pBluescript-directional cloning vector modified by Creative-genomics.The direction will be 5’ end/EcoRI and 3’ end/XhoI.
• Libraries will not be arrayed nor amplified. Creative-genomics will provide transformed cells.
Features and advantages:
• ASH cDNA subtraction does not use a normalization (self-eliminating) step.
• ASH cDNA subtraction is effective for longer cDNA (usually >1.2Kb). cDNA is full-length-enriched and independent of PCR suppression.
• ASH cDNA subtraction provides non-exponential amplification before subtraction because double-stranded cDNA is not required.
• ASH cDNA cloning is directional and allows for construction of expression libraries.
ASH library construction steps:
• Extraction and purification of total RNA.
• Anti-sense strand synthesis of tester (Full-length enriched).
• Sense strand synthesis of driver by APATM (full-length enriched) and labeling.
• Hybridization at a ratio of >500 :1 (driver: tester).
• Removal of double-stranded hybrids.
• PCR amplification of remaining single-stranded tester cDNA.
• Purification and enzyme digestion, ligation and precipitation.
• Transformation and determination of clone size and recombinant rate by PCR.
• Please note that clone array is not included. Transformed cells will be shipped on dry ice. We recommend that cells be arrayed within 3 days following receipt.