Paul.xu at 22:57, 1 November 2019

2019-11-01T22:57:07Z

New page

Cloning of pDB.His.MBP-Int(1185) Purpose: to clone pDB.His.MBP-Int Date: 2012-5-16 to 2012-5- Notebook: Xiaojing Wang Page: 004249 Clone ID: 1185 Experimenters: Xiaojing Wang Results

digest 406 pET28b-Int with Nde1 + Xho1, --insert digest V14 pDB.His.MBP with Nde1 + Xho1---> vector

[[File:1185 i v digest 5.16.2012.JPG|RTENOTITLE]]

Ligate and transform Select 6 clones to mini-prep 1185 plasmids are digested by Nde1 + Xho1

[[File:1185 digest 5.23.2012.JPG|RTENOTITLE]]

1185-1~-6 may be all correct.

Pick 1185-1 to sequence.

== Protein Expression ==

induction, 37 oC 5 hours

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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
| rowspan="1" colspan="4" | 250, 130, 00, 70, 55, 35, 25, 15, 10
|}

[[File:IHF XIS INT Expression screening.jpg|RTENOTITLE]]

=== == ===

10/21/19

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

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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 
| 
|}

[[File:1185 1078 1186 purification.jpg|RTENOTITLE]]

Purification of 1185

expression host: Nico21(DE3)plysN, LB with 1X 5052, 0.5 mM IPTG, 17 oC o/n

[[File:1185 expression truncated.jpg|RTENOTITLE]]

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| 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
| 
| 
| 
|-
| 
| 
| 
| 
|}

11/1/2019

Use Nico21(DE3)pRare2pLysS, 15 ml, induced with 1 mM IPTG, 37 oC 5 hours

cre in Nico21(DE3)pLysN, (from freezer, not freshly transformed) 8L, induce 37 oC 6 hours, 0.15% arabinose., use 50 ml Ni column

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| Lane
| 1
| 
| MW
| Expression
|-
| 1
| 1078
| pET28b-scIHF2, soluble 
| 22.8
| N
|-
| 2
| 406
| pET28b-Int, soluble 
| 42.5
| N
|-
| 3
| 1185
| pDB.His.MBP-Int, soluble 
| 83.7
| Y
|-
| 4
| 2640
| pCold-MBP-(Ent-TEV)-scIHF, soluble 
| 66.9
| N
|-
| 5
| 2640
| pCold-MBP-(Ent-TEV)-scIHF, inclusion body 
| 66.9 
| Y
|-
| 6~12
| 1010 
| Cre,  
| 
| could be low
|-
| 13
| 1078
| pET28b-scIHF2 
| 22.8 
| N
|-
| 14
| 406
| pET28b-Int 
| 42.5 
| N
|-
| M
| 
| PageRuler Plus
| 
| 
|}

[[File:1185 2640 expression.jpg|RTENOTITLE]]
[[Category:Cloning]]

1185-pDB.His.MBP-Int - Revision history

Paul.xu at 22:57, 1 November 2019