Single Stranded Plasmid DNA Isolation Protocol

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Single Stranded Plasmid DNA Isolation Protocol

Date Added: March 02, 2008 07:47:58 PM

Author: plinkadmin

Category: DNA Protocols

Single Stranded Plasmid DNA Isolation Protocol Step by Step Method

1. Take a bacterial colony transformed with phagemid with insert of DNA of interest from an LB plate containing Ampicillin. Use part of the inoculum to make a patch on a LB plate containing Ampicillin and use the rest to inoculate a 2 ml culture of 2X YT Medium. Make sure that a sufficient amount of the colony makes it into the 2X YT Medium.

2. Grow the culture for 2 to 3 hr or until the culture just begins to look turbid. Add 5 ml of a very concentrated (1 X 10¹² phage per ml) stock of helper phage (M13KO7).

3. Grow the culture for 6 hr at 37°C.

4. Add 1.5 ml of this culture to a microcentrifuge tube and centrifuge at 8,000 rpm in a microcentrifuge for 10 min at 4°C to pellet the cells.

5. Pipette the top 1 ml of the supernatant into a microcentrifuge tube containing 500 μl of 13% PEG Solution (see Hint #2). Mix gently.

6. Incubate the tube at room temperature for 15 min (but no longer).

7. Centrifuge the tube at full speed in a microcentrifuge for 10 min at 4°C. Discard the supernatant.

8. Briefly centrifuge in a microcentrifuge to bring down the residual 13% PEG Solution. Remove this residual solution with a drawn-out Pasteur pipette.

9. Repeat Step #8 once to remove the final bit of 13% PEG Solution.

10. Resuspend the phage pellet in 100 μl of TES or TE.

11. Incubate the phage on ice for 10 min.

12. Vortex the tube 3 times.

13. Add 60 μl of Phenol and vortex the tube for 30 sec.

14. Allow the tube to stand for 5 min and vortex again for 30 sec.

15. Centrifuge the tube in a microcentrifuge at full speed for 10 min at room temperature.

16. Remove 80 μl of the aqueous supernatant (the upper phase) and add it to a microfuge tube containing 5 μl of 3 M Sodium Acetate.

17. Add 200 μl of Ethanol and vortex the tube.

18. Incubate the tube for 15 min at room temperature.

19. Pellet the DNA precipitate by centrifugation in a microcentrifuge for 10 min at full speed at 4°C.

20. Remove the supernatant and resuspend the DNA pellet in 70% Ethanol.

21. Repellet the DNA by centrifugation in a microcentrifuge for 10 min at full speed at 4°C.

22. Remove the Ethanol and allow the pellet to dry.

23. Resuspend the DNA pellet in 25 μl of TE and store it at -20°C.

24. Check the plasmid prep by running 5 μl of the DNA solution on a 0.7% Agarose Gel at 100 V (see Protocol on Electrophoresis of DNA on Agarose Gels).

Single Stranded Plasmid DNA Isolation Protocol Buffers

13% PEG Solution		Filter sterilize. 13% (w/v) Polyethylene Glycol (PEG) 8000 1.5 M NaCl

2X YT Medium		5 g/liter NaCl 16 g/liter Tryptone 10 g/liter Yeast Extract Add to 900 ml ddH₂O and shake until the solutes dissolve. Adjust the pH to 7.0 with 5 M NaOH. Bring the total volume to 1 liter with ddH₂O. Autoclave to sterilize.

LB plates with Amp		15 g/liter Agar 5 g/liter NaCl 5 g/liter Yeast Extract When cool, add Ampicillin to a final concentration of 40 μg/ml 1 ml/liter 1 M NaOH Pour plates, Store at 4°C Autoclave. 10 g/liter Ttryptone

3 M Sodium Acetate

70% (v/v) Ethanol

Phenol (CAUTION! see Hint #1)

TES		10 mM NaCl 20 mM Tris-HCl, pH 7.5 0.1 mM EDTA

TE		10 mM Tris-Cl, pH 8.0 1 mM EDTA

Reagents Needed
Tris Ampicillin Ethanol EDTA Yeast Extract Tryptone M13KO7 Helper Phage Sodium Acetate Polyethylene Glycol (PEG) 8,000 Phenol Sodium Chloride Agar Sodium Hydroxide

Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Or add 300 μl of 20% PEG 8000 in 2.5 M NaCl.