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From 2014.igem.org

Protocols / Restriction Enzyme Cleavage (Digest)

Transfer of nucleic acids was enabled by cutting sequences with restriction endonucleases. The enzymes used here identify their recognition site, cut within it, and leave behind single-stranded overhangs (sticky ends). In case no recognition site is present, attachment can be achieved by adding them to the 5´ end of PCR´s oligonucleotides. To have oriented genetic recombination, two endonucleases generating different overhangs were chosen for each cloning step. The reaction mix used here is shown in the following table.

Table 1: Reaction mixes and temperature programs for the restriction enzyme cleavage.

Volume [μl] Compounds of standard PCR 2.00 10 x Fast Digest Green buffer 1.00 enzyme x 1.00 enzyme y - 500 ng DNA ad 20 µl H2O

Cylcer Program Step Temperature [°C] Time [min] Cycle no. 1. 95 2.0 1 2. 95 0.5 35 3. TA 0.5 35 4. 72 60 b/s 35