Why should I perform a positive-control cDNA synthesis reaction?
Why should I perform a positive-control cDNA synthesis reaction?
A positive-control cDNA synthesis reaction, using control RNA included in each SMARTer kit, enables verification of kit performance and components and helps in evaluation of your sample cDNA library.Tips for preparing the control reactions:
Prepare fresh dilutions of the Control RNA. Do not use previously diluted low-concentration RNA samples, since RNA is less stable at low concentrations.
If attempting to use previously diluted Control RNA, analyze its integrity using an Agilent Bioanalyzer 2100.
Prepare Control RNA dilutions in nuclease-free water or Reaction Buffer containing fresh RNase Inhibitor.
Use nuclease-free, nonsticky 1.5-ml tubes.
Avoid pipetting small volumes (1 µl or less). Dilutions of the Control RNA will be more accurate if, after the first 1-µl dilution, subsequent dilutions are performed using larger volumes (4–5 µl) of RNA.
An example of an electropherogram of positive control cDNA synthesized from 100 of pg Control Total RNA generated by a SMARTer Ultra low kit using 15 cycles of PCR. The cDNA spans the expected 400–9,000 bp with a peak at approximately 2,000 bp.