minigene_lib_part1
Homo sapiens
Protocols: The RON minigene was cloned into the pcDNA 3.1 (+) vector (Invitrogen). The minigene construct contains a sequence of the MST1R gene including exon 11 together with the complete flanking introns and the constitutive exons 10 and 12 (chr3: 49,933,098 - 49,933,837, GRCh37/hg19). The RON minigene library comprises of approximately 6,000 minigenes with three to four mutations on average per minigene. E. coli was transformed with a pool of plasmids generated by cloning of a mutated minigene library. Approximately 6,000 of the resulting E. coli transformants were harvested in LB-media. Next, the containing plasmids were extracted using the QIAGEN Plasmid Midi Kit according to the manufacturer's instructions. DNA was extracted from E. coli using the QIAGEN Plasmid Midi Kit according to the manufacturer's instructions. 8.25 ng of plasmid DNA was used as a template for PCR amplification of three amplicons via an emulsion PCR protocol as described by Williams et al., 2006. The same reverse primer was used for each amplicon (5' AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT-TCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA 3') but different forward primer (in 5'-3' direction):1 CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCG-CTCTTCCGATCTNNNNNNNNNNCTATAGGGAGACCCAAGCTT 2 CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCG-CTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG3 CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCG-CTCTTCCGATCTNNNNNNNNNNAGCTGCCAGCACGAGTTCFollowing the PCR amplification, the following steps were performed to clean the water-in-oil emulsion: First, the water phase was extracted by washing twice with 1 ml of water-saturated diethyl ether. Second, residual solvent from the broken emulsion was removed by centrifugation under vacuum for 12 min at 30°C.Finally, PCR products were purified using the Gene Read Size selection kit from QIAGEN according to the maufacturer's instructions.
ENA first public
2018-06-27
ENA last update
2017-11-21
External Id
SAMEA104394719
INSDC center alias
Institute of Molecular Biology (IMB)
INSDC center name
Institute of Molecular Biology (IMB)
INSDC first public
2018-06-27T17:02:54Z
INSDC last update
2017-11-21T14:47:09Z
INSDC status
public
Submitter Id
E-MTAB-6219:minigene_lib_part1
broker name
ArrayExpress
common name
human
genotype
plasmid pool of microgene library containing MST1R constructs
plasmid
amplicon pool 1
sample name
E-MTAB-6219:minigene_lib_part1