Paul.xu at 22:54, 1 November 2019

2019-11-01T22:54:38Z

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== '''Cloning of 1010-pBad24-HTNCre''' ==

'''Purpose:''' Cloning of 1010-pBad24-HTNCre

'''Date: '''2012-2-28

'''Notebook:003752'''

'''Page:'''

'''Clone ID:1010'''

'''Experimenters:''' yingying

== '''Results''' ==

=== subcloning ===

Date:2012-2-28

[[File:1012+1010clone 2-28-2012.JPG|RTENOTITLE]] 

{| cellspacing="1" cellpadding="1" border="1" style="width: 380px; height: 99px;"
|-
| 
| band 3 
| band 4 
|-
| 
| pTriEx-HTNC (Cre) 298 
| pBad24 545 
|-
| rowspan="2" | digest enzymes 

| rowspan="2" |      Nco1 + Xho1 
| rowspan="2" | Nco1 + Sal1 
|}

{| cellspacing="1" cellpadding="1" border="1"
|-
| 
|}

=== Colony Screening ===

Date: 2012-3-1

Make miniprep and digest with  HindIII

[[File:1012+1010digest detect 3-1-2012.JPG|RTENOTITLE]]Purification

clone 2,3,4,6 may be right

 

== '''Result intepretation/Summary''' ==

* 

== '''Conclusion''' ==

== Purification ==

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
| 1178
| 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
| 1178
| pET28b-scIHF2 
| 22.8 
| N
|-
| 14
| 406
| pET28b-Int 
| 42.5 
| N
|-
| M
| 
| PageRuler Plus
| 
| 
|}

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

1010-pBad24-HTNCre - Revision history

Paul.xu at 22:54, 1 November 2019