Team:IPN-UNAM-Mexico/Protocols

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Contents

Competent cells with RbCl

  1. Take 5 ml of liquid SOB and incubate at 37 °C overnight
  2. Take 1 ml of cell culture and innoculate into 500 ml of YENB media and incubate at 37 °C and 200 rpm until O.D.=0.5-0.55
  3. Transfer the cells to 250 ml bottles (must be cold)
  4. Incubate for 30 minutes on ice
  5. Centrifuge at 2500 rpm for 15 min at 4°C.
  6. Resuspend cells in 20 ml of ice-cold RF1 solution
  7. Incubate for 15 min on ice
  8. Centrifuge at 2500 rpm for 15 min at 4°C.
  9. Resuspend cells in 2 ml of ice-cold RF2 solution.
  10. Incubate for 15 min on ice and divide into 200 μl aliquots and store at -70°C

RF1 solution

  • Rubidium chloride....................... 100 mM
  • Manganese chloride tetrahydrate......... 50 mM
  • Potassium Acetate....................... 30 mM
  • Calcium chloride dihydrate.............. 10 mM
  • Gycerol................................. 15 %
  1. Adjust pH to 5.8 with 0.2 M of glacial acetic acid and sterilize by filtration using a 0.22 μm filter


RF2 solution

  • Rubidium chloride....................... 100 mM
  • MOPS.................................... 10 mM
  • Calcium cloride dihydrate............... 75 mM
  • Gycerol................................. 15 %
  1. Adjust pH to 6.8 with 0.2 M of NaOH and sterilize by filtration using a 0.22 μm filter

CTAB miniprep

  1. Take 1.5 ml of cell culture and centrifuge at 15000 rpm for 3 minutes.
  2. Discard liquid phase
  3. Repeat 2 and 3
  4. Resuspend cells with 1 ml of NaCl 1.2 M with vortex
  5. Centrifuge at 13500 rpm for 3 minutes and discard supernatant.
  6. Add 100 μl of sterile water and resuspend with vortex
  7. Add 200 μl of STET and mix with vortex.
  8. Add 4 μl of lysozyme and mix with vortex.
  9. Incubate for reaction to occur for 5 min at 37°C then boil for 45 seconds.
  10. Centrifuge at 15000 rpm for 10 min.
  11. Discard pellet.
  12. Add 8 μl of CTAB and centrifuge at 15000 rpm for 5 min.
  13. Discard supernatant
  14. Resuspend with 300 μl of NaCl 1.2 M using vortex.
  15. Add 1 ml of ethanol and incubate at -20°C for 20 min.
  16. Centrifuge at 15000 rpm for 10 min.
  17. Discard supernatant.
  18. Wash with 1 ml of 70% ethanol and centrifuge for 3 min.
  19. Discard ethanol by decantation.
  20. To dry remaining ethanol use a thermoblock at 65°C.
  21. Resuspend with 100 μl of sterile water and add 1 μl of RNase.
  22. Incubate at 37°C for 30 min.

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