4 protocols
AccessionType
PCR_amplification
DNA ends were blunted using DNA pol, and annealed linkers were ligated to the blunt ends. Subsequently, the DNA was amplified using low cycle numbers to assure linearity of amplification. After Sandman T et al, Nat Prot 1, 2006
specified_biomaterial_action
The polyclonal rabbit antibody against Ttk69 was a generous gift from the Azorin Lab and Travers Lab. For chromatin immune-precipitations, the chromatin was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0.1% SDS / 0.1% sodium deoxycholate, 1mM PMSF) and precleared with 25 ul 50% protein A sepharose beads (Sigma) at 4 degr. for 1 hr. To precipitate the protein of interest, antisera were added to each aliquot of precleared chromatin and incubated at 4 degrs on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 25 ul protein A sepharose at 4 degr. for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with increase NaCl (500 mM), 1x in 250mM LiCl / 10mM Tris-HCl pH 8.0 / 1mM EDTA / 0.5% NP-40 / 0.5% sodium deoxycholate and 2x in TE. The DNA was eluted after protease digest and RNAase treatment by incubation at 65 degree for 6h and phenol chloroform extracted. The DNA fragments were amplified using linker mediated PCR reactions (2x 20 cycles) and purified using Minelute PCR purification columns (Qiagen).
specified_biomaterial_action
For chromatin immune-precipitations, the chromatin was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8.0 / 1mM EDTA / 1% TritonX100 / 0.1% SDS / 0.1% sodium deoxycholate, 1mM PMSF) and precleared with 25 ul 50% protein A sepharose beads (Sigma) at 4 degr. for 1 hr. To precipitate the protein of interest, 1-3 ul of pre-immune (mock) serum were added to each aliquot of precleared chromatin and incubated at 4 degrs on a rotary shaker overnight. The antibody/antigen complexes were precipitated with 25 ul protein A sepharose at 4 degr. for 3 hrs and washed 1x with RIPA buffer, 3x with RIPA buffer with increase NaCl (500 mM), 1x in 250mM LiCl / 10mM Tris-HCl pH 8.0 / 1mM EDTA / 0.5% NP-40 / 0.5% sodium deoxycholate and 2x in TE.
grow
Wildtype Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degrees. Three rounds of 0-1hr prelays were performed to stimulate egg release from the females and thus increase the quality of staged collections. Staged 2 hr populations of embryos were collected and aged at 25 degr for 6 hours to reach the required stage of development. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at RT for 15 minutes. The reaction was terminated with 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. A small number of embryos from each collections was set aside to stage each collection.