Service Procedure

Step 1: Selection of RNA and vector

• Provide us with cells, tissue or RNA. If you wish to use commercially available RNA as a source for your library, please indicate this to us and we will purchase the RNA for you.
• Choose the appropriate library vector

Step 2: Library construction

• If you provide tissue or cells, we will isolate total or polyA+ RNA from it (please see guidelines).
• We perform a stringent quality control on the RNA using Experion lab-on-chip equipment
• 1st strand cDNA is generated by reverse transcription
• cDNA is normalized and is confirmed by qPCR using an abundant marker gene (e.g. actin or GAPDH, for more details please see Normalized Libraries)
  
• The normalized 1st strand cDNA is size-selected and split into two size pools to optimize representation of big and small fragments
• 2nd strand cDNA is generated separately on both size pools
• The two size pools are quality checked using Experion lab-on-chip equipment
• The two size pools are separately ligated into the library vector

Step 3: Quality control

• A test amplification is carried out to optimize amplification parameters
• Quality control is performed. Only cDNA libraries which pass the following criteria are amplified:
• Normalized cDNA sample shows decreased marker gene abundance
• Approx. 1x10⁶ independent clones (minimum complexity 7x10⁵ independent clones)
• More than 85% plasmids containing inserts
• Average insert size at least 1.2 kb

Step 4: Amplification

• The final library is amplified in E. coli
• Primary stocks are generated
• Plasmid DNA is isolated

Step 5: Delivery of the following reagents and data

• Library quality control sheet
• At least 500 µg of purified library plasmid - ready to screen!
• Primary glycerol stocks for later amplification of the library

Ordering information