REMI- Restriction-enzyme-mediated insertional mutagenesis

REMI- Restriction-enzyme-mediated insertional mutagenesis

From Peter Devreotes' web page, based on Adachi et al. 1994.


[MATERIALS] - [INDEX]

Procedure

For 24 REMI's

  1. AX2 cells that have been grown in log phase for 2 days are harvested at a density of 4-6 x 106 cells/ml.

  2. Harvest 4 x 106 cells. Wash 1x in ice cold E-buffer. Resuspend in 10 ml ice cold E-buffer. Keep on ice.

  3. Add 100 mg plasmid (pBSR1 or pBSR3 x BamHI).

  4. Take 4 control samples (no enzyme); 0.4 ml each.

  5. Add 50 units (5 ┬Ál) DpnII to remaining cell suspension (5 units DpnII/ml).

  6. Electroporate at 1.2 kV and 3 mF, using BioRad Gene Pulser with 5 Ohm resistor in series with chamber. Time constant must be 0.5-0.7. Use 0.2 cm cuvettes .

  7. After electroporation incubate10 minutes on ice.

  8. Add 2 ml of Healing solution and transfer to a Petri dish. Let sit 15 minutes.

  9. Add 12 ml HL5 medium and spread the cells evenly in Petri dish.

  10. Next day add 5 mg/ml (6 ml; 10 mg/ml) Blasticidin.

  11. Transformants appear in appr. 5 days.

  12. Screen about 3 times the number of transformants.


Materials

  • E-buffer
    • 10 mM Na/K phosphate buffer pH 6.1
    • 50 mM sucrose

  • Healing solution
    • 100 mM CaCl2
    • 100 mM MgCl2

  • Blasticidin stock
    • 10 mg/ml blasticidin

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