Protocol for DNA base composition analysis of bacterial cells

Protocol for DNA base composition analysis of Bacterial Cells

1.     Take 10 µl DNA solution  and 10 µl  Salmon sperm DNA in eppendrof tube (1 µg/ µl).

2.     Boil in water bath for 5 min. at 100⁰C

3.     Cool rapidly in ice.

4.     Add 10 µl Nuclease-P1

5.     Incubate at 37⁰C for 1 hour.

6.     Then add 10 µl Glycine buffer, 10 µl Alkaline Phosphatase.

7.     Incubate 8 hrs (overnight) at 37⁰C

8.     Add 60 µl sterile 3 D/W water.

9.     Inject 100 µl hydrolysate in the GC vial insert & store at -20⁰C until Chromatographed.

  Reagent for G +C mol % analysis

1.     Nuclease P-1 buffer (for 10 ml):

Ø  0.033g (33mg) Sod. Acetate (40mM)

Ø  0.0057g (5.7mg) ZnSO₄.7H₂O(2mM)

Ø  10 ml 3D/W

Ø  Adjust pH to 5.3 by adding NaoH or acetic acid.

Ø  Store in 4⁰C.

2.     Nuclease P-1 (1mg/ml): (Stock 500U/ml, Required 20U/ml)

Ø  Take 960 µl Nuclease P-1 buffer in eppendrof tube.

Ø  Add 40 µl  Nuclease P-1 (500 U/ml stock).

Ø  Dispense in small aliquots & store in -20⁰C.

3.     10 ml Glycine- Sodium Chloride Stock Solution^#:

Ø  0.77 g Glycine

Ø  0.586 g NaCl

Ø  10 ml 3D/w

Ø  Store in 4⁰C.

4.     Glycine Buffer (10 ml):

Ø  Take 0.63 ml of stock ^#:

Ø  Add 9.37 ml 3D/W

Ø  Adjust pH to 10  by conc. NaOH (1M)

Ø  Autoclave

Ø  Store in 4⁰C.

5.     Alkaline Phosphastase Solution (for 10 ml):

Ø  3.2M (NH₄)₂SO₄   = 4.23 g

Ø  1mM MgCl₂.6H₂O = 2.033mg

(If 1mM MgCl₂ then take 0.9521 mg)

Ø  1mM ZnCl₂ = 1.363 mg

Ø  3D/W = 10 ml

Ø  pH 7.0

Ø  Autoclave and store in 4⁰C.  

6.     Alkaline Phosphatase enzyme (1 ml):

Ø  Take 1 ml alkaline phosphatase solution in mirocentrifuge tube

Ø  Add 1 µl alakaline phosphatase

Ø  Divide in small aliquots (100  µl) per eppedroff.

Ø  Keep in -20⁰C.

7.     Salmon Sperm DNA:

Ø  Take 900 µl 3D/W (Sterile)

Ø  Add 100 µl Salmon Sperm DNA

Ø  Distrubute in a small aliquots

8.     Sample DNA:

Ø  Concentration (1 µg/ µl, i.e 1000 concentration in Nanodrop Measeurement)

HPLC  Requirements:

1.      Eluent: 0.2M ammonium phosphate (pH4.0)

2.      Acetonitrile (40:1 v/v)

3.      Detector: 270 nm

4.      Rentention Time within 30 mins

Peaks of nucleotides