1. Grind 2 g of seedlings into a fine powder in liquid nitrogen using a precooled mortar and pestle. 2. Collect the samples (approximately 5 ml) into a 50 ml tube and resuspend in 5 ml of RG lysis buffer (see Recipe 3). 3. Filter the resulting slurry through four layers of Miraclo...
25–50% sucrose gradient was prepared in 13.2 mL ultracentrifuge tubes (Beckman Coulter, 331372) using Biocomp Gradient Master with the following recipe: 25 or 50% sucrose (w/v), 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide. ...
l IVT reactions using the following recipe: 50 μg ml−1 of DNA template, 14 μg ml−1 of homemade T7 polymerase, 40 U ml−1 of RNase inhibitor (Promega), 6 mM of each NTP, 1 U ml−1 of pyrophosphatase (Thermo Fisher Scientific) and 1× IVT buffer. ...
Cells were harvested by adding lysis buffer (0.15 ml) containing 4% SDS, 20 mm DTT, and 0.1 m Tris, pH 8.6, to each plate. Lysates were incubated at 95 °C for 5 min, sonicated, and then clarified by centrifugation (14,000 × g, 15 min). Proteins were quantified using the micropl...
NTES buffer (see recipe) Refrigerated microcentrifuge Sample preparation 1. Follow steps 1 and 2 in the Basic Protocol. Cell lysis and protein precipitation 2. Add 200 µl NTES buffer and 200 µl of 1:1 phenol:chloroform (4°C). 3. Thaw while shaking vigorously. It is important that...
(2.08 g NaHSO3, 0.67 g ammonium sulfite monohydrate in 5.0 ml 50% ammonium bisulfite) to speed up DNA 5mC sequencing26,27. While attempting to reproduce the experiment, we observed that the mixture prepared according to this recipe required incubation at an elevated temperature to ...
Cell extracts were prepared using cell lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 5 mM sodium vanadate, 1 μg ml−1of aprotinin, leupeptin, and pepstatin)...
The second strand cDNA was generated using the fol- lowing recipe: 10 µl of 5× second strand buffer (500 mM Tris-HCl (pH 7.8), 50 mM MgCl2, 10 mM DTT), 30 nmol dNTPs (Invitrogen, cat. no. 18427-013), 2 U of RNase H (Invitrogen, cat. no. 18021- 014) and 50 U of ...