ena-PROJECT-ISCIII-CNM-18-10-2012-14:26:19:163-11
STUDY OF THE VARIABLE REGION FROM THE HEAVY CHAIN OF THE IMMUNOGLOBULINS EXPRESSED BY THE B-LYMPHOCYTE POPULATIONS CD19+CD45RLO AND CD19+CD45R+
EXPERIMENT was performed by RNA extraction and cDNA conversion of CD19+CD45RLO AND CD19+CD45R+ cells. RT-PCR amplifications were performed with 1 U of FastStart DNA polymerase (Roche Molecular Systems) in a PTC-200 DNA Engine cycler (Bio-Rad). VHDJH-CH-rearranged alleles were amplified by PCR using a VH-degenerated primer and CH-specific primers (for IgM, IgG1 and IgA). The size of the PCR products is around 270-290bp depending on the Immunoglobulin isotype, DH, JH usage and N insertions. The reaction products were cloned using the Dual Promoter TA cloning kit (PCR® II vector, Invitrogen, Life Technologies, San Diego, CA), and transformed into JM109 Escherichia coli competent cells (Promega, Madison, WI). Positive colonies were selected by X-gal/IPTG color selection procedure and were submitted to a PCR amplification reaction. PCR products were cleaned with PCR clean-up kits (Mo Bio Laboratories, Carlsbad, CA) and sequenced with the 5´-primer in an ABI3500 XL analyzer automatic sequencer with the BigDye sequencing mixture (Applied Biosystems, Carlsbad, CA).
EXPERIMENT was performed by RNA extraction and cDNA conversion of CD19+CD45RLO AND CD19+CD45R+ cells. RT-PCR amplifications were performed with 1 U of FastStart DNA polymerase (Roche Molecular Systems) in a PTC-200 DNA Engine cycler (Bio-Rad). VHDJH-CH-rearranged alleles were amplified by PCR using a VH-degenerated primer and CH-specific primers (for IgM, IgG1 and IgA). The size of the PCR products is around 270-290bp depending on the Immunoglobulin isotype, DH, JH usage and N insertions. The reaction products were cloned using the Dual Promoter TA cloning kit (PCR® II vector, Invitrogen, Life Technologies, San Diego, CA), and transformed into JM109 Escherichia coli competent cells (Promega, Madison, WI). Positive colonies were selected by X-gal/IPTG color selection procedure and were submitted to a PCR amplification reaction. PCR products were cleaned with PCR clean-up kits (Mo Bio Laboratories, Carlsbad, CA) and sequenced with the 5´-primer in an ABI3500 XL analyzer automatic sequencer with the BigDye sequencing mixture (Applied Biosystems, Carlsbad, CA).