E2-Crimson template (Clontech) was PCR amplified to add BsiWI (5′) and MluI (3′) restriction sites for cloning purposes using the following conditions: KOD buffer (1×), MgSO4 (1.5 mM), dNTPs (0.2 mM each), forward primer (0.3 μM; GGCCGGCCCGTACGcgtacgGCCACCATGGATAGCACTGAGAACGTCATCAAGCCCTT), reverse primer (0.3 μM; GGCCGGCCacgcgtCTACTGGAACAGGTGGTGGCGGGCCT), and KOD Hot Start DNA Polymerase (0.02 U/μL). KOD PCR reaction used the following cycling conditions: 95°C for 2 minutes; 50 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 70°C for 30 seconds; 60°C for 5 minutes. PCR products were purified (QIAquick PCR Purification Kit) and cloned with Zero Blunt PCR cloning kit. Cloned products and lentiGuide-puro were separately digested with BsiWI (New England Biolabs) and MluI (New England Biolabs) in 1× Buffer 3.1 at 37°C (New England Biolabs). Digest of lentiGuide-Puro (Addgene plasmid ID 52963) was performed to remove the puromycin cassette. Then digested PCR product was ligated into the lentiGuide backbone.
E2 crimson template
Manufactured by Takara Bio
The E2-Crimson template is a fluorescent protein designed for use in molecular biology applications. It is a variant of the E2-Crimson protein, which exhibits bright red fluorescence. The E2-Crimson template can be used as a fluorescent marker or reporter in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using e2 crimson template
1
Cloning of E2-Crimson Template into lentiGuide-Puro
2
Cloning of E2-Crimson Template into lentiGuide-Puro
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E2-Crimson template (Clontech) was PCR amplified to add BsiWI (5′) and MluI (3′) restriction sites for cloning purposes using the following conditions: KOD buffer (1×), MgSO4 (1.5 mM), dNTPs (0.2 mM each), forward primer (0.3 μM; GGCCGGCCCGTACGcgtacgGCCACCATGGATAGCACTGAGAACGTCATCAAGCCCTT), reverse primer (0.3 μM; GGCCGGCCacgcgtCTACTGGAACAGGTGGTGGCGGGCCT), and KOD Hot Start DNA Polymerase (0.02 U/μL). KOD PCR reaction used the following cycling conditions: 95°C for 2 minutes; 50 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 70°C for 30 seconds; 60°C for 5 minutes. PCR products were purified (QIAquick PCR Purification Kit) and cloned with Zero Blunt PCR cloning kit. Cloned products and lentiGuide-puro were separately digested with BsiWI (New England Biolabs) and MluI (New England Biolabs) in 1× Buffer 3.1 at 37°C (New England Biolabs). Digest of lentiGuide-Puro (Addgene plasmid ID 52963) was performed to remove the puromycin cassette. Then digested PCR product was ligated into the lentiGuide backbone.
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