Cloning of 1708-pT7A3-HTNCre

Purpose: Cloning of 1708-pT7A3-HTNCre

Date: 2013-3-22---2013-3-25

Notebook:0

Page:

Clone ID:1708

Experimenters: yingying

Results

Date: 2013-3-25

Make miniprep and digest with Nde1 + Xho1

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.