Difference between revisions of "Team:Arizona State/Experiments"
| Line 9: | Line 9: | ||
<h2> B-Lo </h> | <h2> B-Lo </h> | ||
| − | <p> Insert | + | <h3> Mini-Prep Protocol </h3> |
| + | <h4> Harvest Cell : </h4> | ||
| + | <p> • Pellet 1-5 mL of an overnight recombinant E. coli culture by centrifugation. | ||
| + | o 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 | ||
| + | </p> | ||
<h2> Amber </h2> | <h2> Amber </h2> | ||
Revision as of 17:58, 19 September 2017
