Tissue cDNA, First Strand, Human Adult Normal, Brain, Pituitary, BioGenomics™

Molecular Biology / MB-Cloning-cDNA

-20°C

Supplied as a liquid in 1X RT Buffer (50mM Tris-Cl, pH 8.3, 75mM KCl, 3mM MgCI2, 10mM DTT).

BioGenomics™ Tissue cDNA: cDNA is supplied as First Strand, Multiple Tissue Panels, and Matched Pairs. PCR-ready First Strand cDNA is tissue specific and are ready-to-use for gene discovery or expression analysis. Over 350 cDNAs from human adult and fetal normal tissues, human diseased and tumor tissues, rat, mouse, monkey and plant tissues are included in this extensive collection.

PCR Ready First Strand cDNAs is an excellent source of tissue specific, PCR-ready cDNA, and it can be immediately used for gene discovery or expression analysis. First-Strand cDNA is synthesized from RNA isolated from a wide variety of documented human adult and fetal normal tissues, human diseased and tumor tissues, mouse, rat, monkey and plant tissues. 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 65°C for 10 minutes. The cDNA is in 1X RT buffer. (1X RT Buffer: 50 mM Tris-Cl, pH 8.3, 75mM KCl, 3mM MgCI2, 10mM DTT).

Quality Control:
1. The integrity of the RNA used for cDNA synthesis is examined by visual inspection for the presence of intact bands of 18s and 28s ribosomal RNA when electrophoresed on a denaturing agarose gel. The quality and purity of total RNA were tested by spectrophotometer. A260/280 is between 1.8 and 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 by PCR.
3. The synthesized cDNA was 5’ selected to ensure its full length. The cDNA was used as template for PCR amplification of b-actin gene and an 838bp b-actin band was visualized on 1% agarose gel. b-Actin control primer is included (T5595-0010).

Unit Definition:
1ul cDNA is good for one PCR reaction. The 5' end of human clathrin cDNA (a 6kb gene) has been amplified by PCR from all of the cDNAs.

Features:
Ready to use for PCR
Oligo dT primer used to ensure the entire 3' end of cDNA is present
With some cDNA used as templates, 12kb PCR amplicon was obtained to ensure the intactness of cDNA
The largest selection of cDNAs from different tissues on the market
Documentation of tissues' clinical histories available (additional cost)

Applications:
Immediate PCR Amplification of known genes. Verification of genetic mutation.
Comparison of a specific gene between different tissues. Analysis of mRNA alternative splicing. Gene cloning and target sequencing.

PCR Thermocycling:
• 94°C x 2 minutes, 1 cycle.
• 94°C x 30 seconds, 55°C x 30 seconds, 72°C x 30 seconds, 35 cycles.
• 72°C x 5 minutes, 1 cycle. Then hold at 4°C.

Control PCR Condition:
Ready First Strand cDNA:1.0ul
10X PCR Buffer:2.5ul
10mM dNTP:0.5ul
Control Primers (5uM): 1.0ul
H2O, Nuclease-free:      19.8ul
Taq Polymerase (5u/ul):0.2ul

Storage and Stability:  
Store cDNAs 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.