Appropriate kits

Plasmids used in this study are listed in Table 1. To create pCR2.1-TOPOmini, which lacks the Kan^R cassette, primers TOPO2.1mini_Fwd_NcoI and TOPO2.1mini_Rev_NcoI (Table S4) were used to amplify the backbone of the plasmid pCR2.1-TOPO. The amplified product was cut with NcoI and self-ligated. Standard protocols were used for the isolation of chromosomal DNA, DNA digestion, electrophoresis, and electroporation (Sambrook et al., 1989 ). Enzymes for DNA manipulations were obtained from Promega (Fitchburg, WI), New England Biosciences (Ipswich, MA) or Fermentas (Pittsburg, PA). Plasmid isolations and gel purifications were performed using appropriate kits (Qiagen, Germantown, MD). PCR amplification was performed using ExTaq polymerase, Primestar polymerase (Takara Shuzo, Kyoto, Japan) or PFU Ultra (Agilent Technologies, Madison, WI) and appropriate buffers on 100 ng Xenorhabdus chromosomal template-DNA, 0.2 μM each primer, 0.4 mM dNTPs, and 2.5 U of polymerase. After 2 min incubation at 95°C, 30 cycles of 20 s at 95°C, 30 s at an annealing temperature appropriate for each primer pair, and 60-s kb⁻¹ at 72°C, were conducted, followed by 7 min incubation at 72°C.

DNA from formalin-fixed paraffin-embedded melanoma tissues was extracted using appropriate kits (QIAGEN, Hilden, Germany). DNA from fresh frozen tissues was extracted using the QIAGEN AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN). Concentrations of total DNA extracted from all tumors were measured using an ultraviolet-visible spectrophotometer (NanoDrop Technologies, Wilmington, USA) based on absorption at 260 nm and ratio at 260/280 nm was used as quality control.