LITMUS™ U is a linear vector with 8 nucleotide, 3´non-complementary overhangs that can be used for the rapid cloning of PCR fragments without ligation (Figure 1). LITMUS U has opposing T7 promoters which generate dsRNA from a cloned fragment with little additional sequence by in vitro transcription with T7 RNA Polymerase.
To clone into LITMUS U, PCR products must be generated using primers that have the following sequences at the 5´ end: 5´ GGAGACAUNNN... and 5´ GGGAAAGUNNN... (U = uracil and NNN... = specific primer sequence). After PCR, the DNA product can be directly cloned into LITMUS U using the USER Enzyme (NEB #M5505 ) in a 30 minute reaction without ligation or any purification steps (1) following the USER Friendly Cloning Kit (NEB #E5500) protocol.
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