Pcr dna purification kit

Primers that were used in this study and plasmids used for the mutagenesis and recombinant protein expression are listed in Tables S1 and S2. Isolation and purification of genomic pneumococcal DNA was performed using the QIAGEN Genomic Tip 100/G (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with slight modifications described earlier42 (link). DNA amplifications needed for mutagenesis were carried out by PCR. To amplify pneumococcal DNA by PCR the Taq DNA polymerase (New England Biolabs, Frankfurt, Germany) was used and the reactions were subjected to 30 cycles of denaturation at 94 °C, primer annealing for 30 sec, and elongation at 72 °C. The annealing temperature depended on the primers and extension time on the length of PCR product. For expression cloning the proofreading Pfu polymerase was used as specified by the manufacturer (Stratagene, LaJolla, U.S.). Oligonucleotides were synthesized by Eurofins MWG Operon (Germany). PCR products were purified with the PCR DNA purification kit (Qiagen, Hilden, Germany) and plasmids were isolated and purified with the Qiaprep Spin Midi or Maxiprep Kit (Qiagen, Hilden, Germany). The integrity of the DNA was confirmed by sequencing (Eurofins MWG Operon).

Genomic DNA (200 ng) were used for PCR amplification at 95°C for 45 sec, 60°C for 30 sec, and 72°C for 90 sec for 30 cycles, after an initial denature at 95°C for 5 min. Forward primer was 5’GAA GTA TAA GAT TTT TCA CTC ATA G, and reverse primer was 5’GAA AGG AGA ATC ACT TGA ACC T GGG. PCR products were purified with PCR DNA purification kit (Qiagen, CA) and the sequences were read with a nested primer (5’CAT GCC AAT TGC CTT CTA TG) by a commercial company.

ChIP was conducted as follows (Clontech, cat# 640166). First, cell nuclei were isolated from wild type seedlings, respectively, at the 5th leaf stage cross-linked with formaldehyde, and sonicated to shear the chromatin to fragment with an average size of 0.2–1.0 kb. The samples were incubated with protein A agarose beads (40 μl; 16–157, Millipore). Then, the resulting mixture was incubated at 4°C overnight with anti-H₂B-ubi (Lys 120) combined with protein A agarose beads. The product was washed successively with high-salt NaCl, LiCl, and TE buffers. The washed samples were digested by proteinase K. The DNA fragments were cleaned up using a PCR DNA purification kit (Qiagen). The resulting DNA was analyzed by qRT-PCR using designed primers (Supplementary Table 6). The control was selected using protein A agarose beads incubated with chromatin samples in the absence of anti-H₂B-ubi (Lys 120) antibody. The experiment was repeated three times. OsActin1 was used as negative control.