For the analysis of mutations at IgH Sμ region, genomic DNA was isolated from GC and naïve B cells from immunized mice. Amplifications were performed in 4 independent reactions using the following primers:
(forward) 5’- AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3’; (reverse) 5’- GCGGCCCGGCTCATTCCAGTTCATTACAG-3’. Amplification reactions were carried in a final volume of 25 μl each, using 2.5 U Pfu Ultra HF DNA polymerase (Agilent) for 26 cycles (94°C for 30”, 55°C for 30”, 72°C for 60”). PCR products were purified and fragmented using a sonicator (Covaris), and libraries were prepared according to the manufacturer’s instructions (NEBNext Ultra II DNA Library Prep; New England Biolabs). Sequencing was performed in a HiSeq 2500 platform (Illumina). Analysis was performed as previously described [30 (link)].
(forward) 5’- AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3’; (reverse) 5’- GCGGCCCGGCTCATTCCAGTTCATTACAG-3’. Amplification reactions were carried in a final volume of 25 μl each, using 2.5 U Pfu Ultra HF DNA polymerase (Agilent) for 26 cycles (94°C for 30”, 55°C for 30”, 72°C for 60”). PCR products were purified and fragmented using a sonicator (Covaris), and libraries were prepared according to the manufacturer’s instructions (NEBNext Ultra II DNA Library Prep; New England Biolabs). Sequencing was performed in a HiSeq 2500 platform (Illumina). Analysis was performed as previously described [30 (link)].
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