Materials and Reagents

1. EndoFree Plasmid Kit (QIAGEN, catalog number: 12362 )
   
2. Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
   
3. Sterile saline (0.9% sodium chloride)
   
4. 70% ethanol
   
5. Katemine + Xylzaine mixture (see Recipes)
   

Equipment

1. Micropipettes (Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm, 10 cm length) (Sutter Instruments, catalog number: BF100-78-10 )
   
2. Micropipette puller P-97/ IVF (Sutter Instruments, Novato, CA)
   
3. Electroporator (Electro-Square porator CUY21) (NEPA Gene)
   
4. Platinum plate tweezers-type electrode (Protech International, model: CUY650-P5 )
   
5. Ring forceps (Fine Science Tools, catalog number: 11101-09 )
   
6. Serrated forceps (Fine Science Tools, catalog number: 11000-12 )
   
7. Fine scissors (Fine Science Tools, catalog number: 14060-09 )
   
8. Needle holder (Fine Science Tools, catalog number: 12003-15 )
   
9. Silk Black Braided Suture (Ethicon, catalog number: K871 )
   
10. 3”x3” Sterile Gauze (Dynarex, catalog number: 3353 )
    
11. Square pulse electroporator CUY21 (Nepagene, Japan). A foot pedal is required, because during the surgery both of your hands are occupied to hold the animal and the electrodes, respectively.
    
12. Mouth pipet (Sigma-Aldrich, catalog number: A5177 )
    
13. Fiber optic light Nikon MKII or any other brand)
    
14. Vaporizer for isoflurane anesthetic (Porter Instruments Company, model: 100-F )
    Isoflurane is highly recommended as anesthetics. If this is not available, intraperitoneal injection of the mixture of ketamine (80-100 mg/kg) and xylazine (5-10 mg/kg) will also work. But avoid avertin that is toxic to embryos.
    

Procedure