Phusion u dna polymerase

The library was prepared in two steps: a first multiplex PCR amplification for target enrichment and a second round of amplification with a low number of cycles allowing the barcoding of the template-specific amplicons obtained from the first amplification step. Barcoding was performed using the Nextera™ index kit as previously described [29 (link)]. Locus-specific bisulfite amplicon libraries were generated with tagged primers using Phusion U DNA polymerase (ThermoFisher, cod. F555L).
Amplification products were purified using SPRI-AMPure XT (Agencourt-Beckman Coulter, cod. A63881) quantified with Fluorometer Quantus™ (Promega, cod. E6150) and then employed as template (100 ng) for a second round of PCR (6 cycles). Sample-specific barcode sequences were added in this second PCR. The amplicon library was purified using Agencourt AMPure XP beads (Agencourt-Beckman Coulter, cod. A63881) and then quantitated with the Quantus™ Fluorometer (Promega, cod. E6150). Sequencing was conducted on the MiSEQ (Illumina, cod. 15027617) according to the manufacturer’s protocol. A set of three genes KIF1A, ZAP70, and GP1BB were initially evaluated in parallel by the protocol described by Morandi et al. [15 (link)] using pyrosequencing on GSJunior (Roche) to verify the quantification method in a set of 10 normal donors.

pMevT4 was generated by PstI digestion of pMevT followed by re-ligation of the backbone using T4 DNA ligase and standard molecular biology methods, excising the metabolic pathway cassette (atoB, ERG13, and tHMGR). pMevT-murI RBS variants were generated by uracil-excision cloning of murI into pMevT with a diversity of eight different murI RBS sequences added via the PCR primers for the cloning fragments (Supplementary Table 11). pMVA1 was similarly generated by uracil-excision cloning of PCR fragments, introducing a constitutive J23100 promoter with a PCR primer (Supplementary Table 11). PCRs were conducted by standard procedures with Phusion U DNA polymerase (Thermo). Uracil-excision cloning was performed by approx. equimolar mixing of the respective purified PCR products (Supplementary Table 11) in a 20 μL reaction, including 2 μL FastDigest buffer (Thermo), 0.75 μL USER enzyme (NEB), and 0.75 μL FastDigest DpnI (Thermo). The reaction incubated for 60 min at 37 °C followed by 20 min at 25 °C, and was subsequently transformed into chemically competent E. coli TOP10 cells.