Author: Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek
Title: Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation Document date: 2018_5_17
ID: foskvkwn_20
Snippet: For the analysis of pGKL plasmids, a modified protocol based on that of Pospisek and Palkova (43) was used. Briefly, cells were grown for three days on a dish containing the appropriate antibiotics, transferred into a microplate well and dried for 2 hours at 45°C. After they had dried completely, cells were resuspended in 40 µl of freshly prepared TESP buffer (20 mM Tris-Cl, pH 8; 50 mM EDTA-NaOH, pH 8; 2 % SDS; and 0.5 mg/ml pronase E) and dri.....
Document: For the analysis of pGKL plasmids, a modified protocol based on that of Pospisek and Palkova (43) was used. Briefly, cells were grown for three days on a dish containing the appropriate antibiotics, transferred into a microplate well and dried for 2 hours at 45°C. After they had dried completely, cells were resuspended in 40 µl of freshly prepared TESP buffer (20 mM Tris-Cl, pH 8; 50 mM EDTA-NaOH, pH 8; 2 % SDS; and 0.5 mg/ml pronase E) and dried at 37°C overnight. The sample was completely resuspended the next day in 40 µl of 1x DNA loading buffer (Fermentas). Then, 15 µl of the sample were analyzed using agarose electrophoresis (0.5 % agarose, 1 V/cm) for at least 20 hours. After the electrophoresis, the gel was incubated in a solution containing ethidium bromide (0.5 µg/ml) and RNase A (50 µg/ml) for 3 hours.
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