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.
Making and running a gel
What you need and how much of it to make and run an agarose gel
Materials:
- * 60 ml of TAE liquid into the flask for gel making.
- * 0.6g of the agarose powder to the flask and swirled
- * 6.0 μl of the Syber Safe DNA gel stain to the flask and swirled
Procedure:
How to make gel using ingredients
Gel making/running protocol used:
- Started with adding 60 ml of TAE liquid into the flask for gel making.
- Add 0.6g of the agarose powder to the flask and swirled
- Use the microwave to heat the mixture (1 min, swirl and repeat) till all pieces
dissolved and was a pure clear liquid
- Add 6 μl of the Syber Safe DNA gel stain to the flask and swirled
- Carefully poured the mixture into a blank gel tray with spacers in place.
- Once the gel is cooled, add the KB+ DNA ladder to the first slot, 2nd slot is left
blank and each following slot is to be loaded with 5 μl of each sample.
- Once the slots are filled place the gel (still inside the plastic gel holder) into the
Galileo/ BioRad electric current machine. Run for approximately 45 minutes at
110V.
- To image the gel once finished, use the SynGene gel imager machine.
Chris
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Troubleshoot
Tina
Xylaan
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- Protocols
- Experiments
- Documentation of the development of your project
