cDNA Synthesis System
Optimized one-tube procedure for the synthesis of double-stranded cDNA up to 3 kb from total RNA or mRNA.
For life science research only. Not for use in diagnostic procedures.
- AMV Reverse Transcriptase Buffer, 5x concentrated
- AMV Reverse Transcriptase (25 U/µl)
- 1,4-Dithiothreithol (0.1 M)
- Protector RNase Inhibitor (25 U/µl)
- Oligo (dT)15 primer, 200 µM (1 µg/µl)
- Oligo [(dT24 )T7promotor]65 primer, 100 µM (2 µg/µl)
- dNTP mixture (each nucleotide, 10 mM)
- Control neo mRNA (0.2 µg/µl)
- 2nd strand synthesis buffer, 5x concentrated
- 2nd strand enzyme blend (mixture of DNA Polymerase I, E. coli Ligase and RNase H)
- T4 DNA Polymerase (1 U/µl)
- Double-distilled water
- RNase I (10 U/µl)
- Proteinase K, recombinant, PCR grade (50 U/mll)
Optimized one-tube procedure for the synthesis of double-stranded cDNA from total RNA or mRNA.
The ds cDNA can be used for the construction of non-directional cDNA libraries, as a starting point for substractive hybridization experiments to enrich differentially expressed genes, and for the in vitro transcription of whole cDNA populations to generate labeled cRNA for hybridization on DNA microarrays.
The cDNA Synthesis System uses an optimized one-tube procedure, based on the method of Gubler and Hoffmann.
Sample: 1 - 20 μg total RNA, 0.3 - 2 μg mRNA
Assay time: 60 minutes for 1st strand synthesis, 120 minutes for 2nd strand synthesis
Product: double-stranded cDNA copy of an RNA sample