Haynes Lab:Notebook/Engineering PC-TFs/2013/10/24: Difference between revisions
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#<big>''Prepare''</big>: '''Column Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.''' Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields. | #<big>''Prepare''</big>: '''Column Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.''' Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields. | ||
#'''Load cleared lysate Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.''' | #'''Load cleared lysate Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.''' | ||
# <big> | # <big>''Optional wash''</big> (use only with EndA+ strains). Add 500 µl of the Optional Wash Solution to the column. Centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid. | ||
# <big>''Wash column''</big>: Important Reminder: Verify that ethanol has been added to the bottle of Wash Solution 2. Add 750 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 x g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol. | # <big>''Wash column''</big>: Important Reminder: Verify that ethanol has been added to the bottle of Wash Solution 2. Add 750 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 x g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol. | ||
# <big> | # <big>''Elute DNA''</big>: '''Transfer the column to a fresh collection tube. Add 100 µl of Elution Solution or molecular biology reagent water to the column.''' For DNA sequencing and other enzymatic applications, use water or 5 mM Tris-HCl, pH 8.0, as an eluant. '''Centrifuge at 12,000 x g for 1 minute.''' The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C. Note: If a more concentrated plasmid DNA preparation is required, the elution volume may be reduced to a minimum of 50 µl. | ||
Revision as of 13:55, 25 October 2013
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October 24, 2013Harvest cells: 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.
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