Arizona_State
Protocols
Christina Smith
Vector Digest Protocol for Modular Receiver Vector
Materials
- Vector DNA
- 10x Fast Digest Buffer + Green Dye
- Restriction Enzymes
- BbsI
- SpeI
- Deionized Water
Procedure
- Set up the following reaction using the restriction endonuclease that has the lowest salt concentration in its recommended buffer (total reaction volume 50 µl).
- Set up the following digest reaction on ice:
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| Components |
Volume (µL) |
| dH2O |
25 |
| 10X Fast Digest Buffer + Green Dye |
3 |
| MRV Vector |
10 |
| BbsI |
1 |
| SpeI |
1 |
- Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
- Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
- Incubate
Brianna Lopez
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 Mani
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 Connot
Plate Induction & Imaging Protocol
Introduction
This protocol will lead you through the steps of setting up induction plates for Sender/Receiver combos as well as the imaging of the agar plate.
Materials
- Agar plates
- Sender/receiver bacteria on plates (suggested to be freshly transformed)
- Positive/negative controls (GFP+/-Receiver)
- MicroPipetter (05 ul to 200ul)
- Pipette Tips
- GeneSys Machine
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Procedure
Induction Plate Setup
- Procure agar plates and warm to 37℃.
- Procure plates with desired bacteria (senders,receivers,positive/negative control)
- design/print out template (fig. 1) for induction test
Fig 1: Induction Plate Layout
- Using a micropipettor and a sterile tip, bend the tip on the back of the lid of the agar plate
Fig 2: Bent Tip Method
- Use the bent tip to scrape bacteria from any of the required plates (sender/receiver, pos/neg controls)
- Paint the bacteria (liberally) on the plate in the designated sections.
- Be sure to add enough bacteria so that there is a semi-thick layer that is continuous throughout the designated area.
Imaging the Induction plates
- Put magnetic cover on top of UV light ( will have to adjust placement after imaging)
- Take off Lid of Culture plate
Fig 3: Prepping Culture Plate for imaging
Fig 4: SynGene Settings
- Place culture plate on top of magnetic cover and close the tray. (Fig. 3)
- Machine Settings: (Fig. 4)
- Blots: Fluorescent Blot
- Alexa 488
- Epi Mid Wave UV
- UV06
- Press Green arrow once machine is done calibrating
- Press Capture
- Save as .sgd in order to have the original data still available to be able to save as .tif
- If 3D imaging is needed:
- click edit on the lower right side of the screen and click 3D on following screen (upper left)
- If annotations are needed
- proceed to the edit screen
- click on annotate option in upper left hand corner
- then place text over parts of the image you want to be annotated
- save image in preferred format "as displayed" to save annotations.
Troubleshoot
Christina Smith
Xylaan Livingstone
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