Team:Arizona State/Experiments

• 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 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
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

Completely resuspend the bacterial pellet with 200 µL of the Resuspension Solution.
Vortex or pipette up and down to thoroughly resuspend the cells until homogenous

2. Lyse Cells

Lyse the resuspended cells by adding 200 µL of the Lysis Solution
Mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous (Don’t vortex)
Do not allow the lysis reaction to exceed 5 minutes

3. Neutralize

Precipitate the cell debris by adding 350 µL of the Neutralization/Binding Solution
Gently invert the tube 4-6 times
Pellet the cell debris by centrifuging at ≥ 12,000 x g or maximum speed for 10 minutes
Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out as a cloudy, viscous precipitate.
If the supernatant contains a large amount of floating particulates after centrifugation, re-centrifuge the supernatant

4. Prepare Column

Insert a GenElute Miniprep Binding Column into a provided micro-centrifuge 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

5. 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

6. Wash Column (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
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

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 application, use water or 5 Mm Tris-HCL, Ph 8.0, as an eluant
Centrifuge at ≥ 12,000 x g for 1 minute
Use immediately or store at -20˚C

Team:Arizona State/Experiments

Arizona_State

Experiments

Tina

B-Lo

Mini-Prep Protocol

Harvest Cell :

Amber

Chris

Troubleshoot

Tina

Xylaan

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