Nucelospin gel and pcr clean up kit

Genomic DNA was isolated from each morphology mutant using the Wizard Genomic DNA purification Kit (Promega), and the site of the transposon insertion was identified by the DNA Walking SpeedUp Premix Kit (Seegene Inc, Seoul, Korea) using genomic DNA as template and TS1 and TS2 provided in the kit as primers for Polymerase Chain Reactions (PCRs). All the PCR primers used in this work are listed in S2 Table. The cycling conditions were as described in Deng et al. (2013) [17 (link)]. The resulting PCR fragments were purified using the NuceloSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA), and sequenced using an ABI 3730XL DNA Analyzer (Life Technologies, Carlsbad, CA) at Penn State’s Genomics Core Facility. Sequences were analyzed with the Lasergene (DNAstar, Inc. Madison, WI) software package. The nucleotide sequence of each resulting DNA fragment was compared with the genome sequence of G. hansenii ATCC23769 (GenBank accession number NZ_CM000920.1) [20 (link)] using BLASTN and BLASTtx (blast.ncbi.nlm.nil.gov).

From each cDNA sample, the variable regions of the heavy chain (V_H) were amplified. A rabbit Fc PCR was generated: the forward primer was binding at the beginning of the leader region of VH (ATG​GAG​ACT​GGG​CTG​CGC​TGG​CTT​C) while the reverse primer at the beginning of the constant region of the rabbit heavy chain C_H1 (GGG​AAG​ACT​GAT​GGA​GCC​TTA​GGT​TGC​C). PCR was done using AccuPrime Pfx SuperMix (Thermo Fisher) according to manufacturer’s instructions on a Veriti 96 Well Thermal Cycler (Applied Biosystems). The amount of cDNA template used in each PCR reaction was adjusted according to ddPCR results, in order to provide each reaction mix with the same amount of specific IgG cDNA copies.
PCR conditions for NGS are shown in Table 1.
For each PBMC sample, two technical PCR replicates were made (derived from two cDNA replicates). Each PCR NGS template was generated from a pool of 8 (50 µL each) PCR reactions in order to achieve enough amount of template without raising PCR cycles. After amplification the aliquots were pooled and purified using the NuceloSpin Gel and PCR Clean-up kit (Macherey-Nagel), following the manufacturer’s instructions. DNA was eluted with 30 µL NE buffer.