. Transformation of PL23-40 having a hygromycin

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Hygromycin resistant DX13 aod-2, aod-5, pan-2 AOD2-C-Myc AOD5-N-HA aod-2, aod-5, pan-2 Contains an ectopic copy of aod-5 with N-terminal triple HA tag and ectopic copy of aod-2 with C-terminal triple myc tag 96H9 97B1 1C3 Daod-1, A hygromycin resistant Daod-2, a hygromycin resistant Daod-5, a hygromycin resistantas the microsomal or PMP fraction, whereas the Ight involve appointment supernatant was saved as the cytosolic fraction. Transformation of PL23-40 with a hygromycin resistance plasmid carrying aod-5 with triple HA tag coding sequence inserted right after start off codon This study. Transformation of PL23-40 using a hygromycin resistance plasmid carrying aod-5 with triple myc tag coding sequence inserted prior to quit codon CNA33 PL23-40 This study. Cotransformation of DX13 with two hygromycin resistance plasmids. A single containing aod-2 using a triple myc tag inserted ahead of the stop codon and one particular containing aod-5 having a triple HA tag inserted soon after the commence codon FGSC 18947 FGSC 19465 FGSC 11227 A aod-2, pan-2, a aod-5, pan-2, A aod-2, pan-2, a Contains an ectopic copy of aod-2 with N-terminal triple HA tag. Hygromycin resistant AOD2-C-HA-8 aod-2, pan-2, a Includes an ectopic copy of aod-2 with C-terminal triple HA tag. Hygromycin resistant AOD5-N-HA-1 aod-5, pan-2, A Includes an ectopic copy of aod-5 with N-terminal triple HA tag. Hygromycin resistant AOD5-C-Myc-4 aod-5, pan-2, A Contains an ectopic copy of aod-5 with C-terminal triple myc tag. Hygromycin resistant DX13 aod-2, aod-5, pan-2 AOD2-C-Myc AOD5-N-HA aod-2, aod-5, pan-2 Includes an ectopic copy of aod-5 with N-terminal triple HA tag and ectopic copy of aod-2 with C-terminal triple myc tag 96H9 97B1 1C3 Daod-1, A hygromycin resistant Daod-2, a hygromycin resistant Daod-5, a hygromycin resistantas the microsomal or PMP fraction, whereas the supernatant was saved because the cytosolic fraction. Coimmunoprecipitation To extract nuclear proteins, purified nuclei (100 mg protein) were suspended in 60 ml of suspension buffer [25 mM sucrose, 50 mM Tris-HCl (pH 7.five), 5 mM MgCl2, ten mM CaCl2] and mixed with 60 ml of 0.four M KCl containing 1 mM PMSF and protease inhibitors (final concentrations of 2 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A). The suspension was gently rocked for 2 hr at 4 Insoluble material was removed by centrifugation at 13,000 rpm (16,060 g) for 30 min at 4in a Sorvall Biofuge fresco centrifuge. The supernatant containing salt-extracted proteins was loaded onto a desalting column (Zeba Spin Desalting column; Thermo Scientific, Rockford, IL) which was placed into a fresh 1.five ml Eppendorf tube. The desalting column was centrifuged at 1500 g (4000 rpm) for 2 min at 4in a Sorvall Biofuge fresco centrifuge. The flow-through was straight away employed in coimmunoprecipitation experiments together with the Pierce ProFoundTM HA or c-Myc Tag IP/Co-IP kit (Thermo Scientific). ChIP-seq ChIP-seq was performed on eight separate samples.