1186 pDB.His.MBP-Xis

Cloning of pDB.His.MBP-Xis(1186)
Purpose: to clone pDB.His.MBP-Xis
Date: 2012-5-22 to 2012-5-
Notebook: Xiaojing Wang
Page: 004249
Clone ID: 1186
Experimenters: Xiaojing Wang
Results

digest pET28b-Xis 407 with Nde1 + Xho1-->220 bp insert
digest pDB.His.MBP V14 with Nde1+ Xho1--vector

5/27/2012

digest miniprep with Nde1+ Xho1, clone 1, 4 and 5 maybe ok.

Pick 1186-1 to sequence.

Protein Expression

induction, 37 oC 5 hours

Lane	ID	Name	Pi, MW	Induction	Sample	Expression
1	1708	pET28b-scIHF2			S
2	1203	pET42b-scIHF2			S
3	1074	pET28b-IHFab			S
4	1186	pDB.His.MBP-Xis			S
5	406	pET28b-Int			S
6	1185	pDB.His.MBP-Int
7	1708	pET28b-scIHF2	9.60 / 22.8		P	Y
8	1203	pET42b-scIHF2	8.38 / 54.2		P	Y
9	1074	pET28b-IHFab	9.20 / 25.3		P	N
10	1186	pDB.His.MBP-Xis	6.62 / 52.1		P	Y
11	406	pET28b-Int	9.53 / 42.5		P	N
12	1185	pDB.His.MBP-Int	7.33 / 83.7		P	Y
M		PageRuler Plus	250, 130, 00, 70, 55, 35, 25, 15, 10

==

10/21/19

Purification from 2 L culture. Induce at 18 oC o/n, 0.5 mM IPTG

Lane	Sample, fraction #	Note	Note
1	1185, #37	looks like MBP, truncated, maybe int is not expressed	Cell lysed and regrow,
2	1185, #38	looks like MBP, truncated, maybe int is not expressed	truncated, only MBP
3	1078	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
4	1078	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
5	1186 , #5	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient	has expression
6	1186, #8	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient	has expression
7	1186, #6	washed from Ni using 30 mM Imidazole	has expression
8	1186, #7	washed from Ni using 30 mM Imidazole	has expression
9	1185, #12	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
10	1185, #16	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
11	1185, #17	Ni-->Q, elute using 0.05-->1.5 M NaCl gradient
M	PageRuler Plus	250, 130, 00, 70, 55, 35, 25, 15, 10

Lane	ID	Name	Fraction
1	1186	MBP-Xis	Lot 173
2	1186	MBP-Xis	Lot 172
3	1185	MBP (truncated from Int exp)	Lot 171
4	1185	MBP (truncated from Int exp)	Lot 174  elute from Q-SP, fraction 18
5			Lot 174  elute from Q-SP, F 19
6			Lot 174  elute from Q-SP, F 20
7			Lot 174  elute from Q-SP, F 21
8		junk, discard	Lot 174  elute from Q-SP, F25
9		junk, discard	Lot 174  elute from Q-SP, F26
10			Lot 174 FT on Q-SP F7
11			Lot 174 FT on Q-SP F27
M		PageRuler Plus	250, 130, 00, 70, 55, 35, 25, 15, 10