Haynes Lab:Notebook/Engineering PC-TFs/2013/10/24: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 8: | Line 8: | ||
==October 24, 2013== | ==October 24, 2013== | ||
<big>''Sigma-Aldrich Mini-prep Kit''</big> | <big>''Sigma-Aldrich Mini-prep Kit''</big> | ||
#<big>''Harvest cells''</big>: '''Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation'''. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E.coli culture to a microcentrifuge tube and pellet cells at 12,000 x g for 1 minute. Discard the supernatant. Note: For best results with recombinant E. coli grown in LB (Luria Broth), use 1–3 ml of culture for high copy plasmids or 1–5 ml of culture for low copy plasmids. | |||
# <big>''Resuspend''</big>: cells Important Reminder: Verify that appropriate volume RNase A Solution was added to the Resuspension Solution. '''Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous'''. Incomplete resuspension will result in poor recovery.Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tubes back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 x16 holes.^3 | # <big>''Resuspend''</big>: cells Important Reminder: Verify that appropriate volume RNase A Solution was added to the Resuspension Solution. '''Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous'''. Incomplete resuspension will result in poor recovery.Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tubes back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 x16 holes.^3 | ||
# <big>''Lyse cells''</big>:''' Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous.'''Do not vortex. Harsh mixing will shear genomic DNA, resulting in chromosomal DNA contamination in the final recovered plasmid DNA. Do not allow the lysis reaction to exceed 5 minutes. Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may render it unsuitable for most downstream applications. | # <big>''Lyse cells''</big>:''' Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous.'''Do not vortex. Harsh mixing will shear genomic DNA, resulting in chromosomal DNA contamination in the final recovered plasmid DNA. Do not allow the lysis reaction to exceed 5 minutes. Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may render it unsuitable for most downstream applications. |
Revision as of 13:57, 25 October 2013
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |
October 24, 2013Sigma-Aldrich Mini-prep Kit
|