Pgem t ez

Genomic DNA was isolated from tails of 2–3 cm axolotls using Jetflex Genomic DNA Purification kit (Invitrogen). Following purification of genomic DNA, we used GenomeWalker Universal kit (Clontech) to determine the DNA sequence upstream of the GFAP transcription start site. Two rounds of “walking” were performed using the below primers (5′–3′):
GFAP Promoter GSP1 TTTCCCAACTTCAGCCTCGCAAGACTC
GFAP Promoter NGSP1 ACTTTTGAGGAGGGCCGTTAGAGAAC
GFAP Promoter GSP2 TTCACTGTGGCGTCATGTGGATCGGTAACC
GFAP Promoter NGSP2 TTCCAGCACACTCTGCGTCCCTTTGTTTGC
Distinct PCR bands TA cloned into pGEM-T EZ (Promega) and inserts were sequenced. Based on this analysis we designed primers to amplify ~1.3 kb of the axolotl GFAP promoter using Phusion High Fidelity polymerase (NEB) (5′–3′):
pAxGFAP For 2 XhoI ATACTCGAGTACCTGGCATTGACATTATCTGGTC
pAxGFAP Rev1 HindIII ATAAAGCTTTTTCAGAGTTTCCCAACTTCAGCCT
The resulting PCR product was ligated as a XhoI and HindIII fragment into pGL3 Enhancer luciferase reporter plasmid (Invitrogen).

Primers were designed to clone the Daphnia pulicaria Sir2 ORF using MacVector based on the published D. pulex genome (DappuDraft_290541: wFleaBase) [36 (link)]. We designed four primers such that the two PCR products would cover the entire ORF in two overlapping pieces, the “5′ half” (883 bp) and the “3′ half” (1065 bp) with an overlap of 46 bp (Figure 1B) between the two halves around an internal MscI restriction enzyme site. The primers were designed with a restriction site Nde1 for the 5′ end of the 5′half and BamHI for the 3′ end of the 3′half (sequence of primers is given in the next section). The PCR products were cloned into pGEMT-EZ (Promega) and the sequence of D. pulicaria Sir2 ORF was confirmed and submitted to GenBank (KT960973). We utilized the internal MscI restriction enzyme cut site to join each half of the Sir2 PCR product from their respective 5′half Sir2/pGEMT EZ and 3′half Sir2/pGEMT EZ constructs. A three-piece ligation was performed to join the two halves of the Daphnia Sir2 ORF thereby sub-cloning the full length ORF into the pBSIIKS+ (Stratagene) that contained an in-frame FLAG tag on the amino terminus of Sir2. A final sub-cloning in pcDNA3.1- (Invitrogen) resulted in the FLAG- Sir2/pcDNA 3.1- expression construct used for this study.