D N A ECTRACTION FROM SORGUM PLANT LEAF

LABORATORY REPORT PRESENTED TO

DEPARTMENT OF CHEMICAL AND PROCESS ENGINEERING

SCHOOL OF ENGINEERING MOI UNIVERSITY

MARK .W. WABUSELA CPE/33/09

MR.ARIMI

..

MARCH 2011

GENOMIC DNA EXTRACTION BY CETYLTRIMETHYLAMMONIUM BROMIDE REQUIRED SOLUTIONS AND PREPARATION  2CTAB buffer (50ml)  1.0g CTAB powder (=2%)  5.0 ml 1M Tris-HCl pH 8 (=100mM)  2.0ml 0.5M EDTA (mM)  14.0ml 5M NaCl (=1.4M) Make the solution up to 50ml with water. OTHER SOLUTIONS  Add 4 ml of 2-Mercaptoethanol to 1 ml of 2CTAB buffer required  Chloroform  Isopropanol  1M MgCl2 and 3M NaOAc  70% EtOH  1 TE  3 Eppendorf tubes per sample  Set water bath to 65oC PROTOCOL 1. Place three medium-large sorgum leaves in an Eppendorf tube. 2. Homogenize leaves in the Eppendorf tube. 3. 250 l of 2 CTAB/Mercaptoethanol buffer, keep on ice between samples. 4. Incubate samples in 65oC water bath for one hour. 5. Add 250 l of chloroform. 6. Mix samples by inverting for five minutes at room temperature. 7. Centrifuge for 10 minutes at room temperature (15,000 rpm). 8. Collecy clear upper layer (400 l) in a fresh Eppendorf tube. 9. Add 250 l of isopropanol and invert to mix. 10. Incubate at room temperature for at least 10 minutes.

11. Centrifuge for 10 minutes at room temperature (15,000 rpm). 12. Discard supernatant(use yellow tips). 13. Add 320 l of 1 TE and put samples on ice.