RbCl competent cell

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RbCl Competent Cell Preparation

Contents

Solutions and Supplies

  • sterilized 250 mL centrifuge bottles
  • sterilized 1.5 mL microfuge tubes (at least 50)
  • sterilized 100 mL LB in 250 mL flask
  • filter sterilized 100 mL chilled RF1 (33 mL would be used)
  • filter sterilized 50 mL chilled RF2 (4 mL would be used)
  • 1 LB plate without any antibiotic and 6 LB plates with ampicillin (or other antibiotic) resistance

RF1

Combine for 100 mL: [final]
Rubidium Chloride (RbCl, sigma R2252)1.21 g100 mM
Manganese(II) chloride tetrahydrate (MnCl2·4H2O, sigma M3634)0.99 g50 mM
Potassium acetate0.294 g30 mM
Calcium chloride dihydrate (CaCl2·2H2O)0.148 g10 mM
Glycerol15 g or 12 mL15% wt/vol
  • Adjust final pH to 5.8 using 0.2 M acetic acid (maybe 400 μL for 33 mL). Filter-sterilize.
  • Glacial acetic acid: 1.049 g·cm-3 / 60.05 g·mol-1 = 17.47 M

RF2

Combine for 50 mL: [final]
MOPS (sigma M1254)0.105 g10 mM
Rubidium Chloride (RbCl, sigma R2252)0.06 g10 mM
Calcium chloride dihydrate (CaCl2·2H2O)0.55 g75 mM
Glycerol7.5 g or 6 mL15% wt/vol
  • Adjust final pH to 6.8 using 1 M NaOH (maybe 200 μL for 30 mL). Filter-sterilize.

Cell Preparation Procedure

Day 1

  1. Streak DH5α from frozen glycerol stock on the LB plate.
  2. Incubate at 37 °C, over night.
  3. Prepare sterilized LB.

Day 2

  1. Pick up a single colony from the LB plate.
  2. Inoculate to 3 mL sterilized LB.
  3. Incubate at 37 °C, over night.
  4. 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.
  2. Inoculate 1ml of over night culture to 100 mL of LB in flask.
  3. Monitor OD600 from initial until 0.2 to 0.6. [0.4 - 0.55 optimum]
  4. Transfer culture to centrifuge bottle and chill on ice 10 -15 min.
  5. Pellet cells by centrifugation at 2700 g (4200 rpm in an F14 6x250y rotor) for 10 min at 4 °C.
  6. Decant liquid and stand the bottle in an inverted position for < 1 min.
  7. resuspend in 1/3 original volume (33 mL) chilled RF1 buffer gently (NO VORTEX).
  8. Optimally, resuspend using a 25 mL disposable pipet (RbCl will permanently stain glass pipets).
  9. Continue mixing until cells are evenly resuspended and no clumps are visible.
  10. Incubate cells/RF1 on ice for 15 min.
  11. Pellet cells by centrifugation at 580 g (1950 rpm in an F14 6x250y rotor) for 15 min at 4 °C.
  12. Decant liquid and gently resuspend in 1/25 original volume (4 mL) chilled RF2 buffer.
  13. Incubate cells/RF2 on ice for 15 min.
  14. Get eppendor tubes and box ready.
  15. Aliquot 100 ul each into chilled 1.5 mL eppendorf tubes and freeze on dry ice (or ice).
  16. Store at -80 °C.

Determine transformation efficiency

  1. Dilute control plasmid DNA (known DNA conc) to 1 ng/μL and transform using 1 μL.
  2. Thaw competent cells on ice.
  3. Compare previous lot to current lot .
  4. Combine 1 μL of diluted pDNA and 100 μL competent cells.
  5. Incubate on ice 30-60 min (40 min optimum).
  6. Heat shock at 42 °C for 90 sec, place on ice 5 - 15 min.
  7. Add 900ul LB and incubate at 37°C for 30-60 min (45 min optimum).
  8. Plate 100 μL onto antibiotic plate.
  9. Dilute the culture 10 times and plate 100 μL onto antibiotic plate.
  10. Dilute the culture 100 times and plate 100 μL onto antibiotic plate.
  11. Incubate all the plates at 37 °C, over night.
  12. Count the colonies in the next morning.
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