1710-pT7A3-Nde1

Cloning of 1710-pT7A3-Nde1

Purpose: Cloning of 1710-pT7A3-Nde1

Date: 2013-5-31-----2013-6-3

Notebook:0

Page:

Clone ID:1710

Experimenters: yingying

Results

cloning

Colony Screening

Date: 2013-6-3

Make miniprep and digest with nde1+xho1

Conclusion

Nde1 QC

enzyme dilution buffer: 20 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 7.5

lane	1035,pET28b-Tth-Ligase-K294R,200ng	concentration(mg/ml)	digestion time	company
1	Nde1+BamH1	0.4	1 h	Excellgen
2	Nde1+BamH1	0.6	1 h	Excellgen
3	Nde1+BamH1	0.8	1 h	Excellgen
4	Nde1+BamH1	1.2	1 h	Excellgen
5	Nde1+BamH1	1.5	1 h	Excellgen
6	Nde1+BamH1		1 h	NEB

9/29/2013

Purification of Nde1 using 25 ml heparin HyperD column (XK16).

Dilute partial purified Nde1 with 50 mM Tris-HCl. Load to heparin column.

Elute with 250 ml linear gradient of 0.1 M to 0.6 M NaCl. Nde1 is eluted at 0.4 M NaCl.

Lane	Clone ID	Desc
1	1710	Nde1, Eluted from Heparin column
2	1710	Nde1, Eluted from Heparin column
3	1710	Nde1, Eluted from Heparin column
4	1813	FLP, 50 mM I wash from Ni column
5	1813	FLP, 300 mM I eluted from Ni, diluted
6	1710	Nde1, Eluted from Heparin column
7	1813	FLP, 300 mM I eluted from Ni, diluted
8	1813	FLP, heparin column FT
9	1813	FLP, 50 mM I wash from Ni column
10	1835	MBP-Taq-Muts-6XHis, partial purified from Ni column
11	1813	FLP heparin column elute
M		Fermentas PageRuler Plus
12	1813	FLP heparin column elute
13	1813	FLP heparin column elute

Nde1 QC

control: fastdigest NdeI           mg/ml  

purify 7mg/ml and 0.29mg/ml NdeI with Hepain-Hyperd column, use linear Nacl gradient(0.1-0.6M) to elute protein

NdeI elute out when Nacl concentration is 0.4M-0.51M,

Digest 1ug lambda DNA for 30min using 1ul enzyme.

digest 0.5ug 367plasmid DNA, use 1ul NdeI+0.3ul XbaI+0.3ul KpnI