Team:Wash U/Protocol
From 2009.igem.org
(Difference between revisions)
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- | ==''' | + | =='''Miniprep'''== |
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- | :DNA | + | :DNA Miniprep procedure is used to extract and isolate plasmid DNA from whole bacterial cells. This Miniprep kit was obtained from Sigma-Aldrich and exact solutions and formulas are unavailable (e.g. Wash Solution 1). To get exact ingredients please contact Sigma-Aldrich. |
'''Materials''' | '''Materials''' | ||
* Wash Solution 1 | * Wash Solution 1 | ||
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* Lyse Solution | * Lyse Solution | ||
* Elute solution | * Elute solution | ||
+ | * Resuspension Solution | ||
+ | * RNase A solution | ||
+ | * Lysis Buffer | ||
+ | * Neutralization/Binding Buffer | ||
+ | * GenElute HP Binding Columns | ||
'''Procedures''' | '''Procedures''' | ||
- | # Harvest Cells | + | # Harvest Cells: Pellet 1-5mL of E. coli by centrifugation at 12,000RPM for 1 minute and discard supernatant. |
- | # Resuspend Cells | + | # Resuspend Cells: Resuspend cells with 200uL of the Resuspension Solution containing RNase A solution. |
- | # | + | # Cell Lysis: Lyse the resuspended cells by adding 200uL of Lysis buffer. Immediately mix by inverting the tube 6-8 times (Do Not Vortex). Do Not Allow Lysis to last longer than 5 minutes. |
- | # | + | # Neutralization: Precipitate the cell debris by adding 350uL of the Neutralization/Binding buffer and mix by inverting the tube several times. Centrifuge at 12,000RPM for ten minutes. Cell debris, proteins, lipids, SDS and chromosomal DNA should fall out of solution. |
- | + | # Prepare Column: Insert a GenElute Miniprep Binding Column into a microcentrifuge tube. Add 500uL of Column Preparation solution to each column and centrifuge at 12,000RPM for one minute and discard flow through liquid. | |
- | # Prepare Column: | + | # Load Cleared Lysate: Transfer the cleared lysate from step 4 to the column and centrifuge at 12,000RPM for 1 minute and discard flow through liquid. |
- | # Load Cleared Lysate: | + | # Wash Column with Wash Solution 1: Add 500uL of Wash Solution 1 to the column and centrifuge at 12,000RPM for 1 minute and discard the flow through liquid. |
- | # Wash Column with Wash Solution 1: | + | # Wash Column with Wash Solution 2: Add 750uL of Wash Solution 2 to the column and centrifuge at 12,000RPM for 1 minute and discard the flow through liquid |
- | # Wash Column with Wash Solution 2 | + | |
# Centrifuge at 12,000RPM for 1 minute to remove excess ethanol. | # Centrifuge at 12,000RPM for 1 minute to remove excess ethanol. | ||
- | # Elute DNA: | + | # Elute DNA: Transfer column to fresh collection tube. Add 50uL of Elution Solution to the column. centrifuge at 12,000RPM for 1 minute. DNA is now present in the eluate and ready for immediate use or storage at -20C. |
[[Team:Wash_U/Protocol#Procedures|Back To Top]] | [[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
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== '''Recipes''' == | == '''Recipes''' == | ||
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- | ''' | + | '''Luria Broth (LB) Media''' |
* 10g Tryptone | * 10g Tryptone | ||
* 5g Yeast Extract | * 5g Yeast Extract |
Revision as of 16:50, 9 July 2009