Arizona_State
Experiments
Tina
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B-Lo
Mini-Prep Protocol
Harvest Cell :
• 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.
o For best yields for E. coli grown in LB:
use 1-3 mL of culture for high copy plasmids
use 1-5 mL of culture for low copy plasmids
o for best yields for E. coli grown in TB or 2X YT:
use only 1 mL of culture
1. Resuspend Cells (verify that appropriate volume RNase A solution was added to the Resuspension Solution
a. Completely resuspend the bacterial pellet with 200 µL of the Resuspension Solution.
b. Vortex or pipette up and down to thoroughly resuspend the cells until homogenous
2. Lyse Cells
a. Lyse the resuspended cells by adding 200 µL of the Lysis Solution
b. Mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous (Don’t vortex)
c. Do not allow the lysis reaction to exceed 5 minutes
3. Neutralize
a. Precipitate the cell debris by adding 350 µL of the Neutralization/Binding Solution
b. Gently invert the tube 4-6 times
c. Pellet the cell debris by centrifuging at ≥ 12,000 x g or maximum speed for 10 minutes
d. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out as a cloudy, viscous precipitate.
e. If the supernatant contains a large amount of floating particulates after centrifugation, re-centrifuge the supernatant
4. Prepare Column
a. Insert a GenElute Miniprep Binding Column into a provided micro-centrifuge tube, if not already assembled
b. 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
c. Discard the flow-through liquid
5. Load cleared lysate
a. 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.
b. Discard the flow-through liquid
6. Wash Column (verify that ethanol has been added to the bottle of Wash Solution 2)
a. Add 750 µL of the diluted Wash Solution to the column
b. Centrifuge at ≥ 12,000 x g for 30 seconds to 1 minute
c. Discard the flow-through liquid and centrifuge again at maximum speed for 1-2 minutes without any additional Wash Solution to remove excess ethanol
7. Elute DNA
a. Transfer the column to a fresh collection tube. Add 100 µL of Elution Solution or molecular biology reagent water to the column
b. For DNA sequencing and other enzymatic application, use water or 5 Mm Tris-HCL, Ph 8.0, as an eluant
c. Centrifuge at ≥ 12,000 x g for 1 minute
d. Use immediately or store at -20˚C
Amber
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Chris
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Troubleshoot
Tina
Xylaan
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