Paul.xu at 23:02, 1 October 2013

2013-10-01T23:02:34Z

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== '''Cloning of 1710-pT7A3-Nde1''' ==

'''Purpose:''' Cloning of 1710-pT7A3-Nde1

'''Date: '''2013-5-31-----2013-6-3

'''Notebook:0'''

'''Page:'''

'''Clone ID:1710'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1710 clone 5-30-2013.JPG]] 

=== Colony Screening ===

Date: 2013-6-3

Make miniprep and digest with nde1+xho1 

[[Image:1710+645 digest 6-3-2013.JPG]] 

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

== '''Conclusion''' ==

=== '''Nde1 QC''' ===

'''[[Image:Nde1 QC 7-24-2013.JPG]]'''

enzyme dilution buffer: 20 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA, 50% Glycerol, pH 7.5 

{| width="447" border="1" cellspacing="1" cellpadding="1"
|-
| lane
| 1035,pET28b-Tth-Ligase-K294R,200ng 
| concentration(mg/ml) 
| digestion time 
| company 
|-
| 1
| Nde1+BamH1 
| 0.4 
| 1 h 
| Excellgen 
|-
| 2
| Nde1+BamH1 
| 0.6 
| 1 h 
| Excellgen 
|-
| 3
| Nde1+BamH1 
| 0.8 
| 1 h 
| Excellgen 
|-
| 4
| Nde1+BamH1 
| 1.2 
| 1 h 
| Excellgen 
|-
| 5
| Nde1+BamH1 
| 1.5 
| 1 h 
| Excellgen 
|-
| 6
| Nde1+BamH1 
| 
| 1 h 
| NEB 
|}

 

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" height="336" 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 
|-
| 
| 
| 
|}

 

=== '''Nde1 QC''' ===

[[Image:Compare ndei.JPG]] 

control: fastdigest NdeI           mg/ml   

[[Image:NdeICOMPARE.JPG]] 

purify 7mg/ml and 0.29mg/ml NdeI with Hepain-Hyperd column, use linear Nacl gradient(0.1-0.6M) to elute protein

NdeI elute out when Nacl concentration is 0.4M-0.51M,

Digest 1ug lambda DNA for 30min using 1ul enzyme.

[[Image:Ndei compare.JPG]][[Image:Ndei compare2.JPG]]

digest 0.5ug 367plasmid DNA, use 1ul NdeI+0.3ul XbaI+0.3ul KpnI

[[Image:Ndei compare3.JPG]][[Image:367digest.JPG]]

 

 

[[Category:Cloning]]

1710-pT7A3-Nde1 - Revision history

Paul.xu at 23:02, 1 October 2013