### **Bacterial genomic DNA isolation using CTAB** 

> Version Number: 2
>
> Start Production Date: 8-25-04
>
> Stop Production Date:
>
> Author: William S. and Helene Feil
>
> Reviewed by:
>
> Signature:

# Summary

This scaled up CTAB method can be used to extract large quantities of
large molecular weight DNA from bacteria and other microbes.

# Materials & Reagents

> **[Materials/Reagents/Equipment]{.underline} [Vendor]{.underline}
> [Stock number]{.underline}**

##### [Disposables]{.underline}

> 1.5-mL microcentrifuge tube Eppendorf 22 36 320-4
>
> 50-mL Nalgene Oak Ridge polypropylene centrifuge tube VWR 21010-568
>
> 10-mL pipette Falcon 357551
>
> 1-mL pipette tips MBP 3781

##### [Reagents]{.underline}

> CTAB Sigma H-6269
>
> NaCl Sigma S-3014
>
> TE buffer (10mM tris; 1 mM EDTA, pH 8.0). Ambion 9858
>
> Lysozyme Sigma L-6876
>
> Proteinase K Qiagen 19131
>
> 5M NaCl Ambion 9759
>
> 10% SDS Sigma L-4522
>
> Chloroform Sigma C-2432
>
> IsoAmyl alcohol Sigma I-9392
>
> Phenol Sigma P-4557
>
> Isopropanol VWR PX-1835-14
>
> Ethanol AAPER \-\-\-\-\-\-\-\--
>
> RNAse A (100mg/mL) Qiagen 19101

##### [Equipment]{.underline}

> Hot Plate
>
> 250 mL glass beaker
>
> Magnetic stir rod
>
> Thermometer
>
> Automatic pipette dispenser
>
> Sorval 500 Plus centrifuge (DuPont, Newtown, CT)
>
> 65°C water bath
>
> 37°C incubator

# Procedure

Cell preparation and extraction techniques.

(Modification of CTAB method, Current Protocols in Molecular Biology)

**[1.5ml]{.underline}** **[30ml]{.underline}** **[60ml]{.underline}**

1.  Grow cells in broth and pellet at 10,000 rpm for 5 min or scrape
    from plate.

2.  Transfer bacterial suspension to the appropriate centrifuge tube.

3.  Spin down cells in microfuge or centrifuge at 10,000 rpm for 5
    minute.

4.  Discard the supernatant.

5.  Resuspend cells in TE.

6.  Adjust to OD^600^ ≅ 1.0 with TE buffer (10mM tris; 1 mM EDTA, pH
    8.0)

7.  Transfer given amount of cell suspension to a clean centrifuge tube.
    \-\-\-\-\-\-- 740µl 14.8ml 29.6ml

8.  Add lysozyme (conc. 100mg/ml). Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
    20µl 400µl 800µl

This step is necessary for hard to lyse gram (+) and some gram (--)
bacteria.

9.  Incubate for 5 min. at room temperature.

10. Add 10% SDS. Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
    40µl 800µl 1.6ml

11. Add Proteinase K (10mg/ml). Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
    8µl 160µl 320µl

12. Incubate for 1 hr at 37°C.

13. Add 5 M NaCl. Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
    100µl 2ml 4ml

14. Add CTAB/NaCl (heated to 65°C). Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
    100µl 2ml 4ml

15. Incubate at 65°C for 10 min.

16. Add chloroform:isoamyl alcohol (24:1). Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-- 0.5ml 10ml
    20ml

17. Spin at max speed for 10 min at room temperature.

18. Transfer aqueous phase to clean eppendorf (should not be viscous).

19. Add phenol:chloroform:isoamyl alcohol (25:24:1). Mix well.
    \-\-\-\-\-\-\-\-\-\-\-\-\-\-- 0.5ml 10ml 20ml

20. Spin at max speed for 10 min at room temperature.

21. Transfer aqueous phase and add 0.6 vol isopropanol (-20°C).

(e.g. if 400 μl of aqueous phase is transferred, add 240 μl of
isopropanol. \-\-\-- Add 0.6 of vol. \-\-\--

22. Incubate at room temp for 30 min.

23. Spin at max speed for 15 min.

24. Wash pellet with 70% ethanol, spin at max speed for 5 min.

25. Discard the supernatant and and let pellet dry for 5 -- 10 min at
    room temp.

26. Resuspend in TE plus RNAse (99 μl TE + 1 μl RNAse (10 mg/ml)).
    \-\-\-\-\-\-\-- 20µl 400µl 800µl

27. Transfer to sterile microcentrifuge tubes.

28. Incubate at 37°C for 20 min.

29. Run 1 μl in a 1% agarose gel with concentration standards.

Notes and precautions.

In step 1, do not use too many bacterial cells (an OD^600^ of not more
than 1.2 is recommended), or DNA does not separate well from the
protein.

Most of the time, inverting several times is sufficient to mix well.
Shaking too hard will shear the DNA.

Use any protocol for DNA precipitation, the one in this protocol works
well.

# Reagent/Stock Preparation

[CTAB/NaCl (hexadecyltrimethyl ammonium bromide)]{.underline}

Dissolve 4.1 g NaCl in 80 ml of water and slowly add 10 g CTAB while
heating (≈65°C) and stirring. This takes more than 3 hrs to dissolve
CTAB. Adjust final volume to 100 ml and sterilize by filter or
autoclave.