1624-pCold-6His-GST-(E)-(3C)

Cloning of 1624-pCold-6His-GST-(E)-(3C)

Purpose: Cloning of 1624-pCold-6His-GST-(E)-(3C)

Date: 2013-2-5----2013-2-7

Notebook:0

Page:

Clone ID:1624

Experimenters: yingying

Results

cloning

Digest 1612 pCold-GST-E with Nde1 + Nco1--> vector
ligate to annealed oligos
Nde1-3C-Nco1-T+Nde1-3C-Nco1-B

Colony Screening

Date: 2013-2-7

Make miniprep and digest with Pst1 + BglII

2013-2-27

2013-3-4

two Nhe1 sites

protein purification

lane	1	2	3	4	5	6	7	8	9	10
clone ID	626,pT7A3-Taq_d289	1717, pT7A3-6H-(TEV)-MBP-(3C)	1719, pT7A3-MBP-(E3C)-NRF2	1720,pT7A3-MBP-(E3C)-Keap1	1719, pT7A3-MBP-(E3C)-NRF2, fraction 38	1719, pT7A3-MBP-(E3C)-NRF2, fraction 23	cre, 3.9ug/ul	cre,4/2/2013	1624,pCold-6His-GST-(E)-(3C)
host	Nico21DE3plysN	Nico21DE3plysN	BL21DE3pRare2pLysS	BL21DE3pRare2pLysS	BL21DE3pRare2pLysS	BL21DE3pRare2pLysS			Nico21DE3plysN
induction	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n	0.2 mM IPTG 25 oC o/n			0.2 mM IPTG 25 oC o/n
purification	Ni beads	Ni beads	Ni beads	Ni beads	5ml HisTrap HP	5ml HisTrap HP			Ni beads
MW	61KD/5.97	49KD/5.46	112KD/4.82	115KD/5.57	112KD/4.82	112KD/4.82			33KD/6.0