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.
- 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
Amber
Growing samples in culture tubes for growth curves
This is used for growing up colony samples overnight for use in the plate reader.
Materials:
- 15ml culture tubes
- LB (AMP or CHLOR)
- Grown colonies ready to use on petri dishes
- going to use micropipette to grab one colony at a time and add to LB in culture tubes
Procedure:
Collecting colonies and prepping the culture tubes
**Best to do this in the evening so the cultures don't grow too long**
- Label each culture tube with the contents, date and your name
- Add LB (AMP or CHLOR) to the culture tube. (general rule for how much LB - use about 1/4 the container volume. If tube is 15ml use about 3-4ml LB)
- Take pipette and use a clean tip to collect ONE colony from the plate. Discard the tip INTO the culture tube with the colony on the tip.
- Place the lid on the culture tube (not too tight so there is room for gas exchange)
- Place the culture tubes into the shaking (37F) incubator for overnight growth.
- In the morning you can use the Synergy plate reader to check the OD of the sample and dilute or add fresh LB as needed for the test you are running.
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Chris
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Troubleshoot
Tina
Xylaan
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- Protocols
- Experiments
- Documentation of the development of your project
