Supplementary Methods and Legends
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چکیده
Ig gene rearrangement by Ig complementarity determinig region 3 (CDR3) spectratyping . Total RNA was extracted using RNeasy Minikit (Quiagen) or Nucleo Spin-RNA/protein (Macherey-Nagel) columns and was retrotranscribed with Advantage RT for PCR kit (Clontech). cDNA was amplified with the following fast performance liquid chromatography (FPLC) purified primers (Invitrogen): mu1 forward (FW), CAG CTC AGC AGC CTG ACA TCT; mu2 FW, GAC CTC CAC AGC CTG ACA TCT; mu3 FW, GAC CTT AGT AGA TTG ACA TCT; and muJH reverse (RV), CTY ACC TGA GGA GAC KGT GA. MJH RV primer was labeled at 5’ with 6-carboxy-fluorescein (FAM) fluorophore. Forward primer design was based on immunoglobulin heavy chain (IgVH) sequence available on the ImMunoGeneTics (IGMT) database (http://www.imgt.org/) and took advantage of common sequences existing in the framework 3 (FR3) region of about 55% of IgVH regions. Forward primers were admixed and used in conjunction with the MJH (reverse) primer at a final concentration of 400 nM each. Cycling reaction was as follows: 30 seconds at 94°C, 10 seconds at 54°C, and 5 seconds at 72°C for 35 cycles. PCR products were incubated with 0.5 U T4 DNA polymerase (Invitrogen) for 30 min at 37°C to remove nontemplate nucleotide additions and purified using Montage PCR columns (Millipore, Billerica, MA). PCR products were denatured in 15 ml formamide (Applied Biosystems, Monza, Italy) at 94°C for 5 min and transferred on ice. Products were run on an automated sequenator (3100 Genetic analyzer; Applied Biosystem) and analyzed with Peak Scanner 1.0 software (Applied Biosystems).
منابع مشابه
Supplementary Information 1) Additional Information for Main Figures and Supplementary Methods 2) Supplementary Figure Legends (supplementary Figure S1-s8) 3) Supplementary References
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