Paul.xu at 23:09, 1 October 2013

2013-10-01T23:09:36Z

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== '''Cloning of 1813-pT7A3-HTNFlp-Ecoli''' ==

'''Purpose:''' Cloning of 1813-pT7A3-HTNFlp-Ecoli

'''Date: '''2013-9-11---2013-9-12

'''Notebook:0'''

'''Page:'''

'''Clone ID:1813'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1813clone 9-11-2013.JPG]] 

{| width="610" border="1" cellspacing="1" cellpadding="1"
|-
| Lane 
| 1 
| 2 
| 3 
|-
| 
| 1812,pET28b-HTNFlp-Ecoli 
| 1279,pT7A3 
| 1512,pT7A3-Phi29DNAPol 
|-
| 
| colspan="3" |                      Nco1 + Xho1 
|}

 

=== Colony Screening ===

Date: 2013-9-12

Make miniprep and digest with  Nco1 + Xho1

[[Image:1813digest 9-12-2013.JPG]][[Image:1813digest 9-13-2013.JPG]] 

clone 1,2,3,4,6 right

 

== '''Protein Purification''' ==

9/29/2013 

Purification of Nde1 using 25 ml heparin HyperD column (XK16). 

Dilute partial purified Nde1 with 50 mM Tris-HCl. Load to heparin column. 

Elute with 250 ml linear gradient of 0.1 M to 0.6 M NaCl. Nde1 is eluted at 0.4 M NaCl. 

 

[[Image:Nde1 purification.jpg]] 

 

{| width="305" border="1" cellspacing="1" cellpadding="1"
|-
| Lane 
| Clone ID 
| Desc 
|-
| 1 
| 1710 
| Nde1, Eluted from Heparin column 
|-
| 2 
| 1710 
| Nde1, Eluted from Heparin column 
|-
| 3 
| 1710 
| Nde1, Eluted from Heparin column 
|-
| 4 
| 1813 
| FLP, 50 mM I wash from Ni column 
|-
| 5 
| 1813 
| FLP, 300 mM I eluted from Ni, diluted 
|-
| 6 
| 1710 
| Nde1, Eluted from Heparin column 
|-
| 7 
| 1813 
| FLP, 300 mM I eluted from Ni, diluted 
|-
| 8 
| 1813 
| FLP, heparin column FT 
|-
| 9 
| 1813 
| FLP, 50 mM I wash from Ni column 
|-
| 10 
| 1835 
| MBP-Taq-Muts-6XHis, partial purified from Ni column 
|-
| 11 
| 1813 
| FLP heparin column elute 
|-
| M 
| 
| Fermentas PageRuler Plus 
|-
| 12 
| 1813 
| FLP heparin column elute 
|-
| 13 
| 1813 
| FLP heparin column elute 
|-
| 
| 
| 
|}

 

== '''Conclusion''' ==

[[Category:Cloning]]

1813-pT7A3-HTNFlp-Ecoli - Revision history

Paul.xu at 23:09, 1 October 2013