cDNA Total RNA used for cDNA synthesis is isolated by modified guanidine thiocyanate techniques. 11ug total RNA was primed by an oligo dT primer and reverse transcribed by MMLV reverse transcriptase in 40ul final volume. RT Reaction stopped by heating at 65ºC for 10 minutes. The cDNA is in 1x RT buffer. (1x RT Buffer: 50mM Tris-CI, pH 8.3, 75mM KCI, 3mM MgCI2, 10mM DTT). The estimated cDNA concentration is ~2.5ng/ml. 1ul cDNA is good enough for one PCR reaction.
Donor Information:Human, male 26 years old, normal, brain: cerebellum
Quality Control:1. The integrity of the RNA used for cDNA synthesis is examined by visual inspection for thepresence of intact bands of 18s and 28s ribosomal RNA when electrophoreses on adenaturing agarose gel. The quality and purity of total RNA were tested byspectrophotometer. A260/280 is between 1.8-2.0 (detected in 10mM Tris-Cl, pH 7.5)
The ratio of 28S/18S is 1
2. The RNA used for cDNA synthesis is treated by DNase I, and is tested as DNA free RNA byPCR
3. The synthesized human, animal, and cell line cDNA was 5’ selected to ensure its full length
The cDNA was used as template for PCR amplification of beta-actin gene and an 838 bp beta-actin band was visualized on 1% agarose gel. Beta-actin control primer is included. It is enough for 10 PCR reactions
4. The synthesized plant cDNA was used as template for PCR amplification of chloroplast gene
A 458bp chloroplast band was visualized on 2% agarose gel. Chloroplast control primer isincluded. It is enough for 10 PCR reactions.
Storage and Stability:Store at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Aliquots are stable for 6 months.
