Team:Johns Hopkins/Protocols

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JHU.

Contents

Protocols

Plasmid Extraction

Reagents:

  1. Buffer P1 - Resuspension Buffer
    50mM Tris-Cl, pH 8.0, 10mM EDTA, 100ug/mL RNase A
    Storage condition - 4oC after adding RNase A
    Prep - Dissolve 6.06g Tris base, 3.72g EDTA-2H20 in 800mL dH20. Adjust the pH to 8.0 with HCl.
    Adjust the volume to 1 liter with dH2O. Add 100mg RNase A per liter of P1.
  2. Buffer P2 - Lysis Buffer
    200mM NaOH, 1% SDS
    Storage condition - RT
    Dissolve 8.09g of NaOH pellets in 950mL dH2O, 50mL 20% SDS solution.
    The final volume should be 1 liter.
  3. Buffer N3 - Neutralization Buffer for spin columns.
    Composition unknown
    Storage condition - RT
  4. PB Buffer - Binding Buffer
    Composition Unknown (Proprietary)
    Storage condition - RT
  5. Buffer PE - Wash Buffer
    Composition unknown
    Storage condition - RT

Protocol:

  1. Innoculate 3-4 mL of the E. coli cells in glass tubes with the right selection resistance in LB (Luria Bertani) medium.
  2. Transfer solution to a 2 mL Eppendorf tube and spin down at max speed (~13,000 rpm) for 30 seconds.
  3. Pour out LB medium and repeat until all of the solution from the innoculation tube is pelleted at the bottom of the Eppendorf tube. Each spin down after the first requires one full minute.
  4. Re-suspend the pelleted E. coli in 250 µL Buffer P1 and transfer solution to micro-centrifuge tube provided by the Qiaprep Kit. (This can be the same tube as the 2 mL Eppendorf tube) This step requires a strong vortex in order to fully re-suspend the pelleted cells. May require 3-4 minutes of continuous agitation.
  5. Add 250 µL of Buffer P2 and mix gently by inverting the tube 4-6 times. Be very gentle! Shear stresses can ruin the experiment after the lysis of cells from P2 Buffer.
  6. Add 350 µL of Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
  7. Spin down at max speed for 10 minutes. A white pellet near the bottom and side of the walls will form.
  8. Extract the supernatant and add 500 µL of PB buffer. Place mixture into the Qiaprep spin column.
  9. Centrifuge mixture for 1 minute, max speed.
  10. Remove liquid at bottom.
  11. Add 750 µL of PE Buffer to Qiaprep spin column.
  12. Centrifuge mixture for 1 minute, max speed.
  13. Remove liquid at bottom and centrifuge again for 1 minute at max speed to remove more of the PE buffer.
  14. Transfer Qiaprep spin column to sterile Eppendorf tube and add 50 µL of dH2O and let the spin column equilibrate for 10 minutes with the dH2O.
  15. Spin down column for 1 minute, max speed.
  16. Confirm size with gel electrophoresis with 1% agarose gel and appropriate DNA Ladder. (Remember circular plasmids can become supercoiled and produce smaller apparent sizes on the gel)
  17. Use Nanodrop™ to confirm DNA concentration.

Yeast Transformation

To transform a desired plasmid into a host yeast cell.
Date: 8/25/10 Used to transform ref-GFP

Reagents:

  1. PEG-TE LiOAc
    10 mL 10x TE
    10 mL 1 M LiOAc
    80 mL 50% PEG (3350 mw)
    Mix with stir bar, filter sterilize, store at 4 °C
  2. TE-LiOAc
    10 mL 10x TE
    10 mL 1 M LiOAc
    80 mL H2O
  3. 10x TE
    70 mL Tris Base (1M)
    30 mL 1M Tris-Cl
    20 mL 0.5 M EDTA: pH 8.0
    880 mL diH2O
    Autoclave: 45 min
  4. SsDNA-(Stratagene #201190-81)
    Sonicated Salmon Sperm DNA
    ssDNA: 10mg/mL; 1 mL vial
    Store at -20°C
  5. 1 M Lithium Acetate-(LiOAc: Sigm L-6883)
    102.0 g LiOAc
    Bring to 1L diH2O
    Autoclave 45 min.
  6. 50% PEG-3350-(Sigma P-4338)
    250g PEG to 250 mL warm diH2O
    Bring to 500 mL diH2O
    Filter Sterilize

Protocol

  1. Grow yeast cells to log phase in 5 mL YPD medium overnight @ 30 degrees.
  2. Harvest (5 min, 2000 rpm);aspirate supernatant (sterile tip).
  3. Wash cell pellet 1X with 1mL TE-LiOAc; transfer to eppendorph tubes.
  4. Resuspend in 100 µL of TE-LiOAc.
  5. Add (mix after each):
    2.5µL Fresh Boiled ssDNA (10 mg/mL)
    1µL Mini-Prep Plasmid DNA
    800µL PEG - TE -LiOAc
  6. Incubate @ 30 C; 30 min. Heat Shock 42 C; ~20 minutes.
  7. Harvest cells (30 sec,~14000 rpm), aspirate supernatant.
  8. Wash pellet 1x with 1 mL of YPD.
  9. Resuspend in 100-250 µL of YPD. Plate onto YPD. Incubate plates 2 days at 30 C.
    (Optional: plate onto YPD for 1 day and then replica-plate onto selective medium)

CDRE Optimization

Protocol

A 96 well plate was set up with 2 rows of PMC1 and VCX1 knocked out yeast cells, with the CDRE – RPF plasmid in them. Two rows below that were filled with yeast cells with no knockouts and the CDRE – RFP plasmid inserted in it. We also set aside 8 wells at the bottom as cleaning wells and filled them with ethanol.

The shocking of the cells was carried out using a coaxial electrode system, consisting of 8 sets of gold plated electrodes that fit into the 8 wells of a 96 well plate. An oscilloscope was connected in parallel to the electrode. The shocking was done at 8 V and at 20HZ frequency. The pulses were all sinusoidal.

The cells were shocked for increasing time intervals from 30 seconds to 130 seconds in ten second increments. There was also a 0 second control for every row of cells. Between each shocking the electrodes were first repeatedly dipped in the ethanol wells to clean them and then dipped in a water bath to remove any residual ethanol, then dried using Kim wipes.

After shocking the 96 well plate was put in a shaker at 37C for 8 hours. They were then imaged using a Meta 510 confocal microscope. A 60X oil objective was used and Rodamine and FIT-C filters were applied.

After re suspending any settled cells in the well being tested, 20ul of the cell solution was pipetted onto a slide and a cover slip was placed over it. Some oil was spread on the cover slip and the cells were imaged. At least 2 pictures were taken from each sample.

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