Materials and Reagents

1. Oryza sativa L. ssp japonica, Kitaake, Nipponbare, or other cultivars
   
2. Agrobacterium tumefaciens strain EHA105
   
3. pRGEB31 (Xie and Yang, 2013) (Addgene plasmid 51295 , plasmid map is shown in Figure 2)
   
   
   Figure 2. Schematic illustration of pRGE31 and pRGEB31 vectors. The pRGEB31 vector was used in this protocol.
   
   
4. Bsa I (New England Biolabs, catalog number: R0535S )
   
5. 70%, 100% ethanol
   
6. Alkaline phosphatase, calf intestinal (CIP) (New England Biolabs, catalog number: M0290S )
   
7. T4 DNA ligase (New England Biolabs, catalog number: M0202S )
   
8. T4 polynucleotide kinase (T4 PNK) (New England Biolabs, catalog number: M0201S )
   
9. T7 endonuclease I (T7EI) (New England Biolabs, catalog number: M0302S )
   
10. GoTaq DNA polymerase (Promega Corporation, catalog number: M3001 )
    
11. Phusion high-fidelity polymerase (Thermo Fisher Scientific, catalog number: F530S )
    
12. 5x green GoTaq^® reaction buffer (Promega Corporation, catalog number: M7911 )
    
13. QIAGEN plasmid mini kit (QIAGEN, catalog number: 12123 )
    
14. QIAquick PCR purification kit (QIAGEN, catalog number: 28104 )
    
15. Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H9151 )
    
16. Sodium lauroyl sarcosinate (sarkosyl) (Thermo Fisher Scientific, catalog number: BP235-500 )
    
17. CTAB buffer (see Recipes)
    

Equipment

1. 37 °C water bath
   
2. Thermal cycler
   
3.  DNA electrophoresis apparatus
   
4.  Microcentrifuge
   

Procedure