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Tissue cDNA, First Strand, Human Diseased, Alzheimer's Disease, Brain, Hippocampus, BioGenomics

MBS652008

40 Tests

870 USD

cDNA

Tissue cDNA, First Strand, Human Diseased, Alzheimer's Disease, Brain, Hippocampus, BioGenomics

Tissue cDNA, First Strand Diseased, Alzheimer's Disease, Brain, Hippocampus, BioGenomics

Molecular Biology Grade

Supplied as a liquid in 1 X RT Buffer: 50mM Tris-CI, QH 8.3, 75mM KCl, 3mM MgCl2, lOmM DTT.

2.5ng/ul

Store cDNAs at -20 degree C

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

Quality Control: 1. The Integrity of the RNA used for cDNA syntnesls is examined by' visual inspection for the [2resence of intact bands of 18s and 28s ribosomal RNA when electrophoreses 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 1 OmM Tris-CI, 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 2% agarose gel. b-Actin control primer is included.

Unit: 1 ul cDNA is sufficient for one PCR reaction. Donor Information: Human, male, 88 years old, disease, brain, hippocampus. Clinical Diagnosis: Alzheimer's Disease. Control PCR condition: Ready First Strand eDNA: 1 ul. lOX PCR Buffer: 2.5ul. 10mM dNTP: O.5ul. Control Primers (SuM): 1 ul. H20, Nuclease-free: 19.8ul. Taq Polymerase (5u/ul): 0.2ul. PCR Thermocycling:. • 94 degree C x 2 minutes 1 cycle. • 94 degree C x 30 seconds, 55 degree C x 30 seconds, 72 degree C x 30 seconds, 35 cycles. • 72 degree C x 5 minutes, 1 cycle. Then hold at 4 degree C. Reaction Mixture: . PCR Ready First Strand cDNA: 1 ul. lOx PCR Buffer : 2.5ul. 2.5 mM dNTP: 4ul. Control primers (25uM): 1 ul. H20 Nuclease-free: 16.3ul. Taq Polymerase(5u/ul): 0.2ul . The peR thermocycling: . 94 degree C x 2 minutes 1 cycle. 94 degree C x 30 seconds, 55 degree C x 45 seconds, 72 degree C x 30 seconds, 35 cycles. 72 degree C x 5 minutes, 1 cycle. Then hold at 4 degree C.

Cloning; Cloning-cDNA

Total RNA used for cDNA synthesis is isolated by modified guanidine thiocyanate techniques. 1 1 ug total RNA was primed by an oligo dT pnmer and reverse transcribed by MMLV reverse transcriptase in 40ul tinal volume. RT Reaction stor2ped by heating at 65 degree C for 10 minutes. The cDNA is in 1 X RT buffer. (1 X RT Buffer: 50mM Tris-CI, pH 8:1. 75mM KCl, 3mM MgCl2, 10mM D I I). The estimated cDNA concentration is about 2.5ng/ul. 1 ul cDNA is sufficient for one PCR reaction.

40 Tests

870 USD