Spermidine, solution preparation

Fig. 4 Transmission electron micrograph, (a) T4 DNA/spermidine complex prepared from the 1 1000 solution in molar ratio, and (b) pBR322 DNA/DOGS complex prepared from the 1 5 solution...

After 2 h incubation of the prepared antibody beads with UV-crosslinked extract in a cold room, the beads are washed 4 x with 100 /A RIPA buffer (50 mMTris-HCl pH 7.5, 150 rnMNaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) and lx with genomic DNA lysis buffer (50 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 2.5 mM DTT, 0.5 mM spermidine, 1% Triton X-100). Approximately 300 /(I of PK solution (1 mg/ml proteinase K in genomic DNA lysis buffer and 0.2 U//A RNase inhibitor) is added to the total lysate previously kept on ice and the beads are then incubated at 37° for 30 min. Gently flick the tubes to resuspend the beads every 10 min during the incubation. After removal of the proteinase K solution, 300 /A of RNA extraction solution (4 M guanidine thiocyanate, 0.5% sarkosyl, and 25 mM sodium citrate, pH7) is added to the beads, incubated for 10 min and the supernatant is mixed with 30 fig yeast tRNA (as a carrier) and 30 fil of 3 M sodium acetate. The RNA solution is phenol-chloroform extracted, ethanol-precipitated, and the pellet washed once with 70% ethanol. The dry pellet is used for 1st strand cDNA synthesis, followed by PCR analysis. The removal of proteins... 

Despite considerable biochemical interest in the naturally occurring triamine 3,4-tri, spermidine (31), surprisingly few coordination compounds have been reported. The crystal structure of Cu(3,4-tri)Br2 (five-coordinate, square pyramidal) has been determined678 and mer-Co(3,4-tri)X3 (X = NOz, Cl) have been prepared.679 The triaqua complex resulting from the latter is unstable with respect to spontaneous thermal reduction in acid solution (presumably via Co—N bond rupture) to give Co". 

Z-DNA-inducing ligand. Spermidine is used here as an example. Prepare a solution of spermidine in double-distilled deionized (dd) water at a concentration of 20 mM (i eNote 1). 

The lOx Reaction Buffer for T7 in vitro transcription has to be thawed at room temperature before the experiment set up. The spermidine in this buffer can come out of solution and coprecipitate the DNA if the reaction is prepared on ice. 

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