REMI- Restriction-enzyme-mediated insertional mutagenesis

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

[MATERIALS] - [INDEX]

Procedure

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 µl) 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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