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
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| Peaks of nucleotides |