Expanded high fidelity pcr system

The mutational status of the VH186.2 gene was analyzed as described. Briefly, GC (CD38loFashiB220+) and na.ve (CD38+FasloB220+) B-cells were sorted from spleenocytes of wild type or Stim1&2 floxed/floxed, mb1Cre+ mice 7 days after immunization with 100μg of NP-CFF. Genomic DNA from 104 cell equivalents were subjected to two round of genomic PCR amplification protocol using the Expanded High Fidelity PCR system (Roche, Nutley, NJ) and primers previously described G. Esposito et al., Proc Natl Acad Sci U S A 97, 1166 (Feb 1, 2000). PCR fragments corresponding to the Vh186.2 gene were cloned into the pGEM-T easy (Promega) and sequenced using Sanger sequencing. Sequence analysis was performed with the help of DNAStar Lasergene software.

Total RNA was isolated from feline pancreas (RNeasy Micro Kit, Qiagen) and reverse transcribed into cDNA (Omniscript Reverse Transcription Kit, Qiagen). PCR was carried out with the Expanded High Fidelity PCR System (Roche Applied Science) and a TGradient Thermocycler (Biometra) using various primers sets (Table 2) and feline pancreatic cDNA as the template. Thermal cycling parameters were 94°C for 5 min, followed by 34 cycles of amplifications at 94°C for 30 s, annealing for 30 s, 72°C for 45 s, and 72°C for 7 min as the final elongation step. The annealing temperature used varied depending on the primers used.