Difference between revisions of "Team:Toronto/Experiment-Rubidium Chloride Competent Cells"

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Latest revision as of 06:58, 19 October 2016

Rubidium Chloride Competent Cells · Benchling

Rubidium Chloride Competent Cells

Introduction

Making competence cells with RbCl. Reference: http://openwetware.org/wiki/RbCl_competent_cell#RF1

Materials

  • sterilized 250 mL centrifuge bottles
    • sterilized 1.5 mL microfuge tubes (at least 50)
      • sterilized 100 mL LB in 250 mL flask
        • filter sterilized 50 mL chilled RF1 (33 mL would be used)
          • 0.605 g Rubidium Chloride (100 mM RbCl)
          • 0.495 g Manganese(II) chloride tetrahydrate (50 mM)
          • 0.147 g Potassium acetate (30 mM)
          • 0.074 g Calcium chloride dihydrate (10 mM)
          • 7.5 g or 6 mL Glycerol (15% m/v)
        • filter sterilized 50 mL chilled RF2 (4 mL would be used)
          • 0.105 g MOPS (10 mM)
          • 0.06 g Rubidium Chloride (10 mM)
          • 0.55 g Calcium chloride dihydrate (75 mM)
          • 7.5 g or 6 mL Glycerol (15% m/v)

        Procedure

        • Making RF1 Solution
        1. RF1 Solution (50 ml)
        • 0.605 g Rubidium Chloride (100 mM RbCl)
        • 0.495 g Manganese(II) chloride tetrahydrate (50 mM)
        • 0.147 g Potassium acetate (30 mM)
        • 0.074 g Calcium chloride dihydrate (10 mM)
        • 7.5 g or 6 mL Glycerol (15% m/v)
        1. Adjust final pH to 5.8 using 0.2 M acetic acid (maybe 400 μL for 33 mL). Filter-sterilize.
        1. Glacial acetic acid: 1.049 g·cm-3 / 60.05 g·mol-1 = 17.47 M
        • Making RF2 Solution
        1. RF2 Solution (50 ml)
        • 0.105 g MOPS (10 mM)
        • 0.06 g Rubidium Chloride (10 mM)
        • 0.55 g Calcium chloride dihydrate (75 mM)
        • 7.5 g or 6 mL Glycerol (15% m/v)
        1. Adjust final pH to 6.8 using 1 M NaOH (maybe 200 μL for 30 mL). Filter-sterilize.
        • Day 1
        1. Streak DH5α from frozen glycerol stock on the LB plate.
        1. Incubate at 37 °C, over night.
        1. Prepare sterilized LB.
        • Day 2
        1. Pick up a single colony from the LB plate.
        1. Inoculate to 3 mL sterilized LB.
        1. Incubate at 37 °C, over night.
        1. Put RF1, RF2, centrifuge tube and eppendorf tubes into the 4 °C refrigerator.
        • Day 3
        1. Put RF1, RF2, centrifuge tube and eppendorf tubes on ice.
        1. Inoculate 1ml of over night culture to 100 mL of LB in flask.
        1. Monitor OD600 from initial until 0.2 to 0.6. [0.4 - 0.55 optimum]
        1. Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
        1. Pellet cells by centrifugation at 2700 g (4200 rpm in an F14 6x250y rotor) for 10 min at 4 °C.
        1. Decant liquid and stand the bottle in an inverted position for < 1 min.
        1. resuspend in 1/3 original volume (33 mL) chilled RF1 buffer gently (NO VORTEX).
        1. Optimally, resuspend using a 25 mL disposable pipet (RbCl will permanently stain glass pipets).
        1. Continue mixing until cells are evenly resuspended and no clumps are visible.
        1. Incubate cells/RF1 on ice for 15 min.
        1. Pellet cells by centrifugation at 580 g (1950 rpm in an F14 6x250y rotor) for 15 min at 4 °C.
        1. Decant liquid and gently resuspend in 1/25 original volume (4 mL) chilled RF2 buffer.
        1. Incubate cells/RF2 on ice for 15 min.
        1. Get eppendor tubes and box ready.
        1. Aliquot 100 ul each into chilled 1.5 mL eppendorf tubes and freeze on dry ice (or ice).
        1. Store at -80 °C.



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