Paul.xu at 00:29, 22 May 2013

2013-05-22T00:29:38Z

New page

= '''Cloning of '''1708-pT7A3-HTNCre =

'''Purpose:''' Cloning of 1708-pT7A3-HTNCre

'''Date: '''2013-3-22---2013-3-25

'''Notebook:0'''

'''Page:'''

'''Clone ID:1708'''

'''Experimenters:''' yingying

== '''Results''' ==

=== cloning ===

[[Image:1708 clone 3-21-2013.JPG]] 

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=== Colony Screening ===

Date: 2013-3-25

Make miniprep and digest with  Nde1 + Xho1

[[Image:1708 digest detect 3-25-2013.JPG]] 

 

 

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

* 

== '''Conclusion''' ==

== Protein Purification ==

Protein Purification: 

1. Lyse cells in pH7.5, 50 mM Phosphate buffer with 500 mM NaCl and 1 mM beta-ME. 

2. Load  cleared lysate to 30 ml SP FF, wash with pH7.5, 50 mM Phosphate buffer with 500 mM NaCl and 1 mM beta-ME. 

3. Elute (reverse-flow) with 1 M NaCl in pH7.5, 50 mM Phosphate buffer, 1 mM beta-ME, to 15 ml MEP column 

4. Wash with 10 CV PBS.

5. Elute with (reverse-flow) with NaOAc buffer, pH 5.0, 300 mM Arginine, 1 mM beta-ME.

6. Concentrate and load to TSK gel filtration column

Gel1: 1708 is after MEP column

[[Image:1748_gel-filtration_1676_1728_1708.jpg]] 

gel 2.

1708 Bio-Sil TSK gel filtration fractions 29 to 38 , the last sample before Fermentas PageRuler Plus marker is flow through of MEP column 

 a sharp peak appear in 36~38 (5 ml per fraction), This peak has a samll size protein, do not know what it is.

 

[[Image:Cre_purification_gel_filtration_5_21_2013.jpg]] 

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[[Category:Cloning]]

1708-pT7A3-HTNCre - Revision history

Paul.xu at 00:29, 22 May 2013