The cDNA coding sequences of the first and second cytoplasmic loop domain of human TMEM39A were cloned into the pGBKT7 vector and screened against a normalized universal human cDNA library (Clontech, 630481), following instruction of the Matchmaker Gold Yeast Two-Hybrid System (Clontech, 630489). Verification of positive colonies was achieved by co-transforming wild-type or YR-mutant TMEM39A loop domain (in pGBKT7 Vector) with genes of interest (in pGADT7 Vector) following the instruction of YeastMaker Yeast Transformation System 2 (Clontech, 630439) as well as plasmids from re-cloned cDNA.
Normalized universal human cdna library
Manufactured by Takara Bio
The Normalized Universal Human cDNA Library is a collection of complementary DNA (cDNA) molecules derived from human tissue samples. The library has been normalized to ensure even representation of transcripts, providing a comprehensive and unbiased representation of the human transcriptome.
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Lab products found in correlation
6 protocols using normalized universal human cdna library
1
Yeast Two-Hybrid Screening of TMEM39A
2
Yeast Two-Hybrid Screening of TMEM132D
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The cDNA coding sequence of the C-terminal domain of human TMEM132D was cloned into the pGBKT7 vector and screened with a normalized universal human cDNA library (Clontech, 630,481) in pGADT7 Vector, following instructions in the Matchmaker® Gold Yeast Two-Hybrid System (Clontech, 630,489). Verification of positive colonies was achieved by co-transformation of extracted bait and prey plasmids following the instruction of YeastMaker™ Yeast Transformation System 2 (Clontech, 630,439) and by bait/prey plasmids with re-cloned cDNA.
3
Yeast Two-Hybrid Screening of TMEM131
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The cDNA coding sequences of the PapD-L domain of C. elegans TMEM131 and the C-terminal TRAPID of human TMEM131 were cloned into the pGBKT7 vector and screened with a normalized universal human cDNA library (Clontech, 630481), following instructions in the Matchmaker Gold Yeast Two-Hybrid System (Clontech, 630489). Verification of positive colonies was achieved by cotransforming PapD-L domains from different species (in pGBKT7 vector) and genes of interest (in pGADT7 vector) following the instruction of the Yeastmaker Yeast Transformation System 2 (Clontech, 630439), as well as plasmids from recloned cDNA.
4
TMEM39A Cytoplasmic Loop Interactors
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The cDNA coding sequences of the first and second cytoplasmic loop domain of human TMEM39A were cloned into the pGBKT7 vector and screened against a normalized universal human cDNA library (Clontech, 630481), following instruction of the Matchmaker® Gold Yeast Two-Hybrid System (Clontech, 630489). Verification of positive colonies was achieved by co-transforming wild-type or YR-mutant TMEM39A loop domain (in pGBKT7 Vector) with genes of interest (in pGADT7 Vector) following the instruction of YeastMaker™ Yeast Transformation System 2 (Clontech, 630439) as well as plasmids from re-cloned cDNA.
5
Yeast Two-Hybrid Screening of MCAM Interactors
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The Matchmaker Gold yeast two-hybrid system (Clontech) was used according to the manufacturers' instructions. The yeast strain Y2HGold, which expresses bait protein from the plasmids of pGBKT7-MCAM-1, -2 or -3, was used to screen the pre-made universal human (normalized) cDNA library (Clontech) in the Y187 yeast strain using a yeast two-hybrid method. Coding sequences of the human FGF2, FGF4, FGF8, PLC-γ1, PLC-γ2 or IP3R1 cDNAs were screened from the human cDNA library and confirmed by the sequencing method. The primers for construction of pGBKT7-MCAM-1 (AA, 24-322), pGBKT7-MCAM-2 (AA, 323-560) and pGBKT7-MCAM-3 (AA, 583-646) are listed as follows (lower cases indicate added adaptor sequences). MCAM-1 forward, 5′-gccgaattcATGGTGCCCGGAGAGGCTGAGC-3′; and MCAM-1 reverse, 5′-ggatcccCTACTGACATTCATAGCGCCCAC-3′. MCAM-2 forward, 5′-gccgaattcATGGGCCTGGACTTGGACACCA-3′; and MCAM-2 reverse, 5′-ggatcccCTAGCCCCGGCTCTCCGGCTCC-3′. MCAM-3 forward, 5′-gccgaattcATGAAGAAGGGCAAGCTGCCGT-3′; and MCAM-3 reverse, 5′-ggatcccCTAATGCCTCAGATCGATGTAT-3′.
6
Yeast Two-Hybrid Screening of SK2b
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The yeast two-hybrid screen, using full-length human SK2b as bait, was performed using the Matchmaker Gold Gal4 Two-Hybrid System (Clontech) and a universal human normalized cDNA library (Clontech) according to the manufacturer’s instructions, and as previously described [48 (link)].
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