User:NYOUSHA Yousefi/Notebook/Protein Engineering/2013/02/05

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Procedure 1)
(Objective)
Line 10: Line 10:
==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
+
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==  

Revision as of 19:30, 18 February 2013

Project name Main project page
Previous entry      Next entry

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 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
  4. These samples were stored at -80 degree C freezer


Personal tools