User:NYOUSHA Yousefi/Notebook/Protein Engineering/2013/02/05: Difference between revisions
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==Objective== | ==Objective== | ||
The wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, | 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 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) | ||
==Objective 2== | ==Objective 2== | ||
Extracting bacteria cells | |||
==Procedure 2== | ==Procedure 2== | ||
# 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 | |||
# 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 | |||
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Revision as of 17:32, 18 February 2013
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TitleHb buffer exchange ObjectiveThe wild type Hb's buffer was exchanged to a new buffer containing 1g manitol, 0.5g PEG, and 50ml phosphate pH 7 Procedure 1
Objective 2Extracting bacteria cells Procedure 2
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