Clontech pcr select cdna subtraction kit

SMART amplification and SSH were carried out using SMARTer™ PCR cDNA Synthesis Kit and Clontech PCR-Select™ cDNA Subtraction Kit (Clontech Laboratories, Inc, CA, USA) according to the manufacturer’s instructions. Control mouse liver total RNA (the positive group), deionized H₂O (the negative group) and the primers (3′ SMART CDS Primer II A and 5′ PCR Primer II A) were provided by the kit. Briefly, 3.5 μl (50 ng) total RNA extracted from two-cell embryos of the test and control groups were successively subjected to first-strand cDNA synthesis, amplified by long-distance PCR, purified by column chromatography, digested with RsaI, diluted to a final concentration of 300 ng/μl in 1X TNE buffer, and subjected to adaptor ligation.
SSH materials consisted of cDNA from the test two-cell embryos as the tester and cDNA from the control two-cell embryos as the driver for forward subtraction, and vice versa for reverse subtraction. After the first and second hybridizations, hybridized samples 1 and 2 were mixed and then incubated at 68 °C overnight, followed by a primary PCR and a secondary PCR. After an agarose/ethidium bromide gel analysis, the products were used for DNA ligation. The same procedures listed above were also performed for reverse subtraction and control subtraction.