Sybr green qpcr supermix udg w rox kit

Chromatin immunoprecipitation was performed as described (31 ). Briefly, yeast cultures were treated with formaldehyde (1% final) for 15 min before glycine (0.32% final) was added. The cells were washed twice in Tris-buffered saline (TBS) buffer, once in FA lysis buffer (pH 7.5, 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate and 0.1% (w/v) sodium dodecylsulphate [SDS]), spun down and frozen at −80°C. Cell pellets were lysed in FA lysis buffer (with 2 mM phenylmethylsulfonyl fluoride, PMSF) by bead beating using a Precellys 24 homogenizer (Precellys). The cell lysates were incubated with anti-HA (ab9110, Abcam) or anti-Myc (ab32, Abcam) antibodies together with protein G Dynabeads (Invitrogen) on a wheel at 4°C overnight. The immunoprecipitates were washed five times in FA lysis buffer and eluted in ChIP elution buffer (50 mM Tris·Cl, pH 7.5, 10 mM EDTA and 1% (w/v) SDS). The samples were treated with proteinase K and incubated overnight at 62°C to reverse the crosslinks. Immunoprecipitated DNA was purified using PCR purification kit (QIAGEN) and quantified using the SYBR Green qPCR SuperMIX-UDG w/ROX kit (11744500, Invitrogen) by a SteponePlus qPCR machine (Life Technologies; all samples were also normalized by input DNA, quantified on the same plates).

ChIP was performed as previously described with the minor modification that formaldehyde crosslinking was performed for 60 min (47 ). 3 μg of mouse anti-FLAG (Sigma F3165) or goat anti-mouse (Dako P0447) antibodies were used per sample. Immunoprecipitated DNA was quantified by qPCR using a SYBR Green qPCR SuperMIX-UDG w/ROX kit (Invitrogen).