1720-pT7A3-MBP-(E3C)-Keap1

Cloning of 1720-pT7A3-MBP-(E3C)-Keap1

Purpose: Cloning of 1519-pT7A3-T3-DNA-Ligase

Date: 2013-4-13---2013-4-16

Notebook:0

Page:

Clone ID:1720

Experimenters: yingying

Results

cloning

lane5: pcr

lane6:vector:1699 pT7A3-MBP-(E3C)

Nde1 + Xho1

Colony Screening

Date: 2013-4-16

Make miniprep and digest with Nde1 + Xho1

protein screening

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

Protein expression of KEAP1 clones

clone ID	expression	ge image
1625,pT7A3-MBP-(3c)-KEAP1	yes	1625 1626 1656
1656,pCold-GST-(3c)-KEAP1	need to use GST beads	1625 1626 1656
1676,pCold-MBP-(3C)-KEAP1	yes	1661 1674 1683
1701,pMAL-c5E-KEAP1	yes
1720,pT7A3-MBP-(E3C)-Keap1	no	626 1717 1719 1720

Cloning of 1720-pT7A3-MBP-(E3C)-Keap1

Purpose: Cloning of 1519-pT7A3-T3-DNA-Ligase

Date: 2013-7-26---

Notebook:0

Page:

Clone ID:1720

Experimenters: yingying

Results

cloning

lane	1	2
	1720,pT7A3-MBP-(E3C)-Keap1(old clone)	1718 pT7A3-MBP-(3C)-MCS-(TEV)-6H
enzymes	Nde1 + Xho1

Colony Screening

Date: 2013-7-28

Make miniprep

digest with BglII

transform BL21star(DE3)pRare2pLysS with clone #4

Date: 2013-7-29

digest with Nde1 + Xho1