Difference between revisions of "User:NYOUSHA Yousefi/Notebook/Protein Engineering/2013/02/05"

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
 
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
 
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==Objective==
 
==Objective==
The wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, o.5g PEG, and 50ml phosphate pH 7
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The wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, 0.5g PEG, and 50ml phosphate pH 7
  
 
==Procedure 1==  
 
==Procedure 1==  
# Column chromatogrophy was rinsed with 25ml of distilled water and 25ml of new buffer solution containing 0.5g PEG, 1g manitol,and 50ml of phosphate buffer, pH 8  
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# Column chromatogrophy was rinsed with 25ml of distilled water and 25ml of the new buffer solution containing 0.5g PEG, 1g manitol,and 50ml of phosphate buffer, pH 8  
 
# 2.5ml of WT Hb and 2.5ml of the new buffer were added to the column and this 5ml was collected in a new tube (2% manitol, 1% PEG, WT Hb, pH 7 phosphate)
 
# 2.5ml of WT Hb and 2.5ml of the new buffer were added to the column and this 5ml was collected in a new tube (2% manitol, 1% PEG, WT Hb, pH 7 phosphate)
  
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# The bacteria expressing Mn and Ni were allowed to grow in media for 6 hours after addition of IPTG, which is a signal that stimulates protein expression
 
# The bacteria expressing Mn and Ni were allowed to grow in media for 6 hours after addition of IPTG, which is a signal that stimulates protein expression
 
# The media was divided and balanced into four smaller flasks and they were placed in centrifuge  at 4500rpm, 4 degree C for 20 min
 
# The media was divided and balanced into four smaller flasks and they were placed in centrifuge  at 4500rpm, 4 degree C for 20 min
# After 20 min, the supernatant of the samples was discarded and the pellets including WT Ni and Mn Hb, BL-21(DE3) were resuspended in Tris pH 9 buffer
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# After 20 min, the supernatant of the samples was discarded and the pellets including WT Ni and Mn Hb, BL-21(DE3) were resuspended in Tris pH 9 buffer separatley
 
# These samples were stored at -80 degree C freezer       
 
# These samples were stored at -80 degree C freezer       
  

Latest revision as of 22:28, 26 September 2017

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Title

Hb buffer exchange

Objective

The wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, 0.5g PEG, and 50ml phosphate pH 7

Procedure 1

  1. Column chromatogrophy was rinsed with 25ml of distilled water and 25ml of the new buffer solution containing 0.5g PEG, 1g manitol,and 50ml of phosphate buffer, pH 8
  2. 2.5ml of WT Hb and 2.5ml of the new buffer were added to the column and this 5ml was collected in a new tube (2% manitol, 1% PEG, WT Hb, pH 7 phosphate)

Objective 2

Extracting bacteria cells

Procedure 2

  1. The bacteria expressing Mn and Ni were allowed to grow in media for 6 hours after addition of IPTG, which is a signal that stimulates protein expression
  2. The media was divided and balanced into four smaller flasks and they were placed in centrifuge at 4500rpm, 4 degree C for 20 min
  3. After 20 min, the supernatant of the samples was discarded and the pellets including WT Ni and Mn Hb, BL-21(DE3) were resuspended in Tris pH 9 buffer separatley
  4. These samples were stored at -80 degree C freezer