Plasmid?DNA?minipreps

There are many methods for DNA preparation without using kits.Below are a few minipreps.These protocols may be scale up to midi or maxi prep if necessary.

1.Ammonium Acetate Method

1)Spin down cell and resuspend in 0.2 ml Solution 1.

2)Lyse cell by adding 0.4 ml solution 2.Leave for 5 min,on ice.

3)Add 0.3 ml 7.5M amm.acetate,spin to pellet cell debris after 5 minutes on ice.

4)Add 0.6 vol isopropanol (0.54 ml) to supernatant,incubate 10 minutes and spin 10-15 minutes.

5)Dissolve pellet in 0.1 ml 2M amm.acetate,Spin after 5 min.on ice to pellet proteins.

6)Add equal volume of isopropanol to supernatant,and spin for 10 minutes to precipitate DNA.

7)Add 50 ul of RNase solution to dissolve DNA.Incubate at 37 °C for 10 minutes.

8)Precipitate DNA by adding 1/2 vol. of 7.5M amm.acetate and 3 vol.isopropanol.Wash pellet with 80% ethanol.

9)Air-dry and resuspend DNA in TE buffer.

Solution 1:50 mM Glucose;10 mM EDTA;25 ml Tris HCl pH8.0.
      Solution 2:1ml 10% SDS;0.2 ml 10M NaOH;8.8 ml water.
      TE buffer:10 mM Tris HCl pH8;1 mM EDTA.
      RNase solution: 50 ul of 10 mg/ml RNase;5 ml TE. 

2.Alternative ammonium acetate method

This is a shorter version of the above.If the DNA is not clean enough. add a step at the end.

Pellet and resuspend 1.5 ml of o/n culture in 200 µl of buffer A;Add 400 ul of buffer B,mix by inversion,incubate on ice for 5 min.Add 300 ul of 7.5 M amm.acetate,mix and incubate on ice for 10 min.Spin at full speed 10 min.Transfer supernatant into new tube with 500 #181;l isopropanol and mixed.Spin 10-15 min.Discard supernatant and wash pellet with 1 ml of 100% EtOH.dry pellets and resuspend in 50 µl H2O or TE.

Buffer A:50 mM TrisHCl pH 8.0,10 mM EDTA,100 ug/ml RNase A.
      Buffer B:200 mM NaOH,1% SDS.
      7.5 M NH4Ac (57.75 g - made up to 100 ml in water) - prepared fresh monthly as ammonium acetate decomposes.

3.Note

1) The oldest plasmid isolation method is by Clewell and Helinski (1969,Proc.Natl.Acad.Sci.USA,62,1159-1166) which is a version of the Triton lysis,and also a gentle method for preparation of large DNA,but the chromosomal clot can only be pelleted at high speeds.

2) EndA strains (e.g. DH5-alpha) is said to be unsuitable for boiling preps (a myth?).

3) Overnight culture can sometimes produce catenae (interconnected supercoiled circles),especially with very high copy number plasmids like bluescript.If catenae is a problem or if you wish to get highest proportion of only formI-DNA,harvest cell at OD=1 (1.5 maximum).

4) To get only supercoiled DNA,purify your DNA over a CsCl gradient in the presence of EtBr.Your supercoiled DNA forms a band well separated from relaxed or linear DNA.Two consecutive gradients are better than one.

5) Lysis buffer containing NaOH should be prepared fresh - NaOH reacts with CO2 from air.

6) The LiCl method can also be used.The RNase,however,is not really necessary for this prep,as most of the RNA goes into pellet but some low MW RNA may remain.

7) Alkaline lysis methods can denature plasmid DNA which make some DNA hard to digest. Therefore avoid leaving the solution in the alkaline state for too long at room temperature. Alternative methods of lysis can be used if it is a problem (e.g. triton, boiling).

8) Other miniprep methods are available at Mark Strom's protocols site.See also "Wicked-Quick" Mini-prep,Merlin Minipreps and others.