Difference between revisions of "Preparing the pBAD and Lysis plasmids for gel electrophoresis"
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− | #Take 1.5µl of the culture in an | + | #Take 1.5µl of the culture in an Eppendorff tube. |
− | # Centrifuge the culture @ 13200 rpm for 3 minutes, making sure the centrifuge is balanced. The centrifugal force created in the centrifuge forces the bacteria in the solution to collect in a pellet at the bottom of the | + | # Centrifuge the culture @ 13200 rpm for 3 minutes, making sure the centrifuge is balanced. The centrifugal force created in the centrifuge forces the bacteria in the solution to collect in a pellet at the bottom of the Eppendorff tube. |
#Repeat process to get required quantity of the cultures i.e. (1.5 µl twice)3.0 µl and centrifuge again. | #Repeat process to get required quantity of the cultures i.e. (1.5 µl twice)3.0 µl and centrifuge again. | ||
Line 7: | Line 7: | ||
#The liquid around the collected pellet, called the supernatant can be discarded. | #The liquid around the collected pellet, called the supernatant can be discarded. | ||
− | #Dissolve the pellet in | + | #Dissolve the pellet in 200µl of [[ALS I]] previously stored at 4 degrees Celsius. |
+ | |||
+ | #Incubate at room temperature(RT) for 3-5 minutes. Incubating at RT enables the DNA to get denatured. | ||
+ | |||
+ | #Add 200µl of [[ALS II]] - mix properly using a Vortex Mixer and incubate at RT for 5 minutes. | ||
+ | |||
+ | #Add 200µl of chilled [[ALS III]], mix properly and incubate the tubes in ice for 10 minutes. | ||
+ | |||
+ | #Centrifuge the tubes @ 13.2k rpm for 15 minutes. | ||
+ | |||
+ | #Take out the aqueous phase and transfer into a fresh Eppendorf tube. | ||
+ | |||
+ | # | ||
+ | |||
... | ... |
Revision as of 07:51, 12 June 2009
- Take 1.5µl of the culture in an Eppendorff tube.
- Centrifuge the culture @ 13200 rpm for 3 minutes, making sure the centrifuge is balanced. The centrifugal force created in the centrifuge forces the bacteria in the solution to collect in a pellet at the bottom of the Eppendorff tube.
- Repeat process to get required quantity of the cultures i.e. (1.5 µl twice)3.0 µl and centrifuge again.
- The liquid around the collected pellet, called the supernatant can be discarded.
- Dissolve the pellet in 200µl of ALS I previously stored at 4 degrees Celsius.
- Incubate at room temperature(RT) for 3-5 minutes. Incubating at RT enables the DNA to get denatured.
- Add 200µl of ALS II - mix properly using a Vortex Mixer and incubate at RT for 5 minutes.
- Add 200µl of chilled ALS III, mix properly and incubate the tubes in ice for 10 minutes.
- Centrifuge the tubes @ 13.2k rpm for 15 minutes.
- Take out the aqueous phase and transfer into a fresh Eppendorf tube.
...