|cDNA synthesis with Time Saver Kit (Amersham)|
Usually double stranded cDNA synthesis is used for such complicated application as library construction or full size gene cloning or subtractive hybridization. If you'll start to combine all the components separately it's very simple to make a mistake which can become really expencive if you use mRNA for your syntesis. I have tried several different systems and found that Time Saver kit from Pharmacia can be the most optimal. Reaction components are premixed in 2 tubes for 1-st and 2-nd strand synthesis reactions. Columns for size selection of obtained cDNA are also provided. You can download the manual from the company web site to get a feel of the procedure. I used the kit with oligo-dT and gene specific primers. In both cases cDNAs up to 6-8 kb were obtained. Time required for the complete procedure is about 3 h. All incubation steps were performed in PCR cycler.
I also tried to use the columns for size separaton of obained products but surprisingly the losses of cDNA were extremely high. I didn't measure them directly, but inderect estimation suggest something like 90-95% losses! Due to the valuability of the material I used I didn't try to opimize the conditions. Nevertheless I would recommend this kit for any application where double stranded cDNA is required.
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