ASH cDNA subtracted library

New! ASH is APA-based, full-length-enriched, single-stranded cDNA subtraction hybridization library.

This unique offering will be offered at an introductory price
 for a limited time. Please inquire for more details.

Also offering a reverse subtraction library for an additional fee. Please contact us to inquire.

ASH library specifications

 
  1. Insert sizes of about 1200bp (based on colony PCR_ with 10% of clones <500bp, and a >95% recombinant rate.
  2. Number of clones: >30,000 for a subtracted cDNA library. Additional clone available upon request for an extra fee.
  3. Full-length enriched.
  4. Directional cloning: Subtracted cDNA will be ligated directionally in pBluescript-directional cloning vector modified by Bio S&T. The direction will be 5’-end/EcoRI and 3’-end/XhoI.
  5. Libraries will not be arrayed nor amplified. Bio S&T will provide transformed cells.
ASH library construction steps

 
  1. Extraction and purification of total RNA.
  2. Anti-sense strand synthesis of tester (Full-length enriched).
  3. Sense strand synthesis of driver by APATM (full-length enriched) and labeling.
  4. Hybridization at a ratio of >500 :1 (driver: tester).
  5. Removal of double-stranded hybrids.
  6. PCR amplification of remaining single-stranded tester cDNA.
  7. Purification and enzyme digestion, ligation and precipitation.
  8. Transformation and determination of clone size and recombinant rate by PCR.
  9. 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.
  10. Library must be stored at -80 degrees.

Major advantages of Bio S&T’s ASH technique versus SSH subtraction method:

ASH cDNA subtraction is full-length-enriched and independent of PCR suppression. SSH involves complete digestion of cDNA which yields small insert size (average size usually <700) and is dependent on PCR suppression.
 



ASH cloning is directional. SSH is non-directional.
 

ASH method does not involve normalization. SSH method is tester-tester, driver-driver hybridization which influences representation of differentially-expressed genes.
 


ASH method provides non-exponential amplification before subtraction because double-stranded cDNA is not required. Prior to hybridization, SSH usually has a PCR amplifyication step to obtain sufficient cDNA for enzyme digestion and adaptor ligation. This can change the cDNA representation before subtraction thereby reducing the subtraction effect.
 




ASH method has a better subtraction effect because 1) a high ratio of driver:tester can be used, 2) normalization not involved and 3) representation well preserved before subtraction hybridization.
 




Please email our Project Manager, Ms. Areti Karadimos, at areti@biost.com with project requirements. A quote will be provided in less than 24 hours.