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Supporting Online Materials Materials and Methods
 

Summary: 1
Supporting Online Materials
Materials and Methods
Plasmid Constructs
Oligonucleotide primers were designed to amplify (from human genomic DNA) a mini-
gene that contains exons 7, 8, and 9 of the adenosine deaminase (ADAR2) gene and
exons 11, 12, and 13 of the putative glucosyltransferase gene (PGT). Each primer
contained an additional extension encoding a restriction enzyme sequence. The PCR
product of ADAR2 and PGT (2.2kb and 3kb, respectively) was restriction digested and
inserted between the KpnI/BglII sites in the pEGFP-C1 vector (Clonthech). The hSlu7
cDNA was a kind gift from Robin Reed and was inserted as described above into pEGFP-
C1.
Site-Directed Mutagenesis
Oligonucleotide primers containing the desired mutations were used to amplify a
mutation-containing replica of the wild type mini-gene plasmid. The PCR products were
treated with 12U DpnI restriction enzyme (New England Biolabs) for 1hr at 37o
C. 1-3Ál
of the DNA was transformed into E.coli DH5 strain, followed by colony-picking mini-
prep and midi-prep extraction (GIBCO/BRL). All plasmids were confirmed by
sequencing.

  

Source: Ast, Gil - Department of Molecular Genetics and Biochemistry, Tel Aviv University

 

Collections: Biology and Medicine