Shreffler:Notebook/retinoic acid trans signaling/2009/05/20: Difference between revisions

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*'''[[User:Neisha A. Rivers|Neisha A. Rivers]] 15:02, 1 June 2009 (EDT)''':
*'''[[User:Neisha A. Rivers|Neisha A. Rivers]] 15:02, 1 June 2009 (EDT)''':


Plasmid Purification:
Plasmid Purification: as per [[Shreffler:Plasmid_Purification|protocol]]


( Note: This process is used for rapid purification of transfection grade plasmid DNA)
( Note: This process is used for rapid purification of transfection grade plasmid DNA)




Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit
----


A) Bacterial culture, harvest, and lysis
Measure the Concentration of DNA using a UV Specrophotometer:
 
( Note: Info on using Specrophotometery) [http://www.icampus.ucl.ac.be/courses/SBIM2520/document/genemol/index/Machines/spectrophitachi/DNAconcentration.html]
 
Sample 1x = 189.7 ng/ul ~ 171 mg of DNA
Ratio = 260/280 = 1.69
 
Sample 2x = 112.3 ng/ul
Ratio = 260/280 = 1.8
 
 
----
 
Prepare Glycerol Stock of Bacteria Culture:
 
1. Vortex the cell culture in 15 ml tube
 
2. Prepare a 20% glycerol solution (80% water + 20% glycerol)
 
3. Take 400 ul of glycerol solution and place it into a 1.5 ml tube)
 
4. Add 600 ul of the cell culture, invert mixture up and down, then place in -80 degree freezer for storage


1. Pellet 150 ml overnight LB culture at 6000 x g for 15 min at 4 degree C


2. Homogeneously resuspend the bacterial pellet in 10 ml Buffer P1


3. Add 10 ml Buffer P2, mis throughly by vigoorously inverting 4-6 times, and incubate at room
temperature for 5 min


4.




Revision as of 14:13, 3 June 2009

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Plasmid Purification: as per protocol

( Note: This process is used for rapid purification of transfection grade plasmid DNA)


Measure the Concentration of DNA using a UV Specrophotometer:

( Note: Info on using Specrophotometery) [1]

Sample 1x = 189.7 ng/ul ~ 171 mg of DNA Ratio = 260/280 = 1.69

Sample 2x = 112.3 ng/ul Ratio = 260/280 = 1.8


Prepare Glycerol Stock of Bacteria Culture:

1. Vortex the cell culture in 15 ml tube

2. Prepare a 20% glycerol solution (80% water + 20% glycerol)

3. Take 400 ul of glycerol solution and place it into a 1.5 ml tube)

4. Add 600 ul of the cell culture, invert mixture up and down, then place in -80 degree freezer for storage





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