1708-pT7A3-HTNCre
Cloning of 1708-pT7A3-HTNCre
[edit]Purpose: Cloning of 1708-pT7A3-HTNCre
Date: 2013-3-22---2013-3-25
Notebook:0
Page:
Clone ID:1708
Experimenters: yingying
Results
[edit]cloning
[edit]
Colony Screening
[edit]Date: 2013-3-25
Make miniprep and digest with Nde1 + Xho1
Result intepretation/Summary
[edit]Conclusion
[edit]Protein Purification
[edit]Protein Purification:
1. Lyse cells in pH7.5, 50 mM Phosphate buffer with 500 mM NaCl and 1 mM beta-ME.
2. Load cleared lysate to 30 ml SP FF, wash with pH7.5, 50 mM Phosphate buffer with 500 mM NaCl and 1 mM beta-ME.
3. Elute (reverse-flow) with 1 M NaCl in pH7.5, 50 mM Phosphate buffer, 1 mM beta-ME, to 15 ml MEP column
4. Wash with 10 CV PBS.
5. Elute with (reverse-flow) with NaOAc buffer, pH 5.0, 300 mM Arginine, 1 mM beta-ME.
6. Concentrate and load to TSK gel filtration column
Gel1: 1708 is after MEP column
gel 2.
1708 Bio-Sil TSK gel filtration fractions 29 to 38 , the last sample before Fermentas PageRuler Plus marker is flow through of MEP column
a sharp peak appear in 36~38 (5 ml per fraction), This peak has a samll size protein, do not know what it is.
