Team:Wash U/Protocol
From 2009.igem.org
Line 233: | Line 233: | ||
=='''Genomic DNA Purification'''== | =='''Genomic DNA Purification'''== | ||
<font size="2"> | <font size="2"> | ||
- | : | + | :Characterization and modeling of individuals parts of our system will require the isolation and purification of genomic DNA from ''Rhodobacter sphaeroides''. One of the main goals of the project is to make BioBricks out of ompR, ompF, pucB/A and the puc promoter coding regions. This DNA however is in chromosomal, not plasmid, form and will require a separate protocol to obtain. Note: This protocol and materials were obtained in a kit called DNeasy from Qiagen. To obtain exact recipes and formulas please contact them. |
'''Materials''' | '''Materials''' | ||
- | + | * Overnight cultures | |
+ | * Buffered ATL | ||
+ | * Proteinase K | ||
+ | * Buffered AL | ||
+ | * Ethanol | ||
+ | * DNeasy mini column | ||
+ | * Buffer AW1 | ||
+ | * Buffer AW2 | ||
+ | * Buffer AE | ||
+ | * | ||
'''Procedures''' | '''Procedures''' | ||
- | + | # Harvest cells by taking 1.5mL from overnight cultures and spinning them down to a pellet. | |
+ | # Resuspend pellets in Buffered ATL | ||
+ | # Add 20uL Proteinase K, mix by vortexing and incubate at 55C for 3 hours shaking every 20-30 minutes | ||
+ | # Vortex 15 seconds and then add 20uL Buffer AL to the samples mixing thoroughly and incubate at 70C for 10 minutes | ||
+ | # Add 200uL ethanol to the sample and mix by vortexing | ||
+ | # Pipet mixture from step 4 into DNeasy mini column sitting in a collection tube. Centrifuge at 12,000RPM for 1 minute and discard the flow through in the collection tube. | ||
+ | # Place the mini column in a new collection tube and add 500 uL of Buffer AW1. Centrifuge at 12,000RPM for 1 minute and discard the flow through and collection tube. | ||
+ | # Place the mini column in a new collection tube and add 500 uL of Buffer AW2. Centrifuge at 12,000RPM for 3 minutes and discard the flow through and collection tube. | ||
+ | # Place the mini column in a clean collection tube and pipet 200 uL Buffer AE on the DNeasy membrane. Incubate at room temperature for 1 minute and then centrifuge for 1 minute at 12,000RPM. | ||
+ | # Repeat elution as described in step 9 with same collection tube to combine both elutes. | ||
<br>[[Team:Wash_U/Protocol#Procedures|Back To Top]] | <br>[[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
Revision as of 19:09, 9 July 2009