BamHI NEB #R0136

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	The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

Protocol tips

The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

Publication protocol

The full-length RasGRP2 cDNA (NCBI accession number NM_153819.1) was amplified by PCR using specific primers (forward primer, 5′-AATaagcttGCAGGCACCCTGGACCTGGAC-3′; reverse primer, 3′-AATggatccTTACAAGTGGATGTCAAACACCC-5′; HindIII and BamHI sites, indicated by lowercase letters, were incorporated to facilitate cloning) and subcloned into a pEGFP-C3 plasmid (BD Biosciences Clontech), generating pEGFP-C3-RasGRP2 with an N-terminal GFP tag. The full-length cDNA clone of RasGRP2 served as a template to generate the recombinant C1 domain. The C1 domain of RasGRP2 was subcloned into the pEGFP-C3 vector using the BamHI and HindIII sites (forward primer, 5′-CATaagcttCACAACTTCCAGGAGAGCAAC-3′; reverse primer, 3′-AATggatccTCAACACTCAACTGACAGGCG-5′). The DNA fragments of the PCR were purified with the QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) and afterward digested with HindIII and BamHI (New England Biolabs, Beverly, MA). After an additional step of purification, the fragments were finally ligated into the GFP-containing pEGFP-C3 plasmid using the restriction sites. The integrity of the inserts was verified by DNA sequencing, which was performed by the DNA Minicore (Center for Cancer Research, National Cancer Institute, National Institutes of Health).

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