G+C content determination

Purified and good quality of genomic DNA is needed to analyze DNA G+C content. The DNA G+C content was determined by reversed-phase HPLC followed by the method of Mesbah et al., 1989. The detailed procedure for DNA G +C mol% is as follows:

II.9.1 Materials          - Genomic DNA of strains and control (Salmon DNA).
          - Incubator
          - Sterilized eppendorfs
          - Sterilized tips and pipettes
          - Centrifuge
          - Water bath
          - Thermometer
          - HPLC

II.9.2 Chemicals-          Nuclease P1
-          Calf Intestinal Phosphatase (CIP)
-          Nuclease P1 buffer
-          Glycine buffer
-          Alkaline phosphatase
-          Molecular biology grade water, autoclaved

II.9.3 Protocols for buffer

a)      Nuclease P1 buffer (pH 5.3)
-          40 mM Sodium acetate
-          2 mM ZnSO₄b)      Glycine buffer (pH 10)
-         Add 63.0 mL of stock glycine-sodium chloride solution to 850 mL of deionized water
-          Adjust pH to 10.0 with 10 M NaOH
-          Dilute to 1 L with deionized water
-          Refrigerate when not in use and prepare fresh weekly
c)      Glycine-sodium chloride stock solution
-          Dissolve 77.4 g glycine and 58.6 g NaCl in deionized water
-          Dilute to 1 L with deionized water and mix
-          Keep refrigerated
d)      Alkaline phosphatase buffer
-          3.2 M (NH4)₂SO4 (pH 7.0)
-          Stabilized by 1 mM MgCl₂ and 1 mM ZnCl₂
                       II.9.4 Procedure-          Heat 10 μL genomic DNA solution (1μg/μL) in 100ºC
-          Cool rapidly into ice
-          Add 10 μL nuclease P1 (20U/mL), mix well
-          Incubate at 37ºC for 2 hours
-          Add 10 μL glycine buffer and 10 μL alkaline phosphatase (1U/mL)
-          Incubate at 37ºC overnight (8 hours)
 Then, samples were centrifuged and water is added to make 100 μL final volumes. The samples were analyzed by HPLC and G + C mol % were calculated as described by Mesbah et al., 1989.