DWA LAB
Home
Lab Manual
biological reagents
Cell Free Systems
DNA
In Vivo Systems
misc methods
overexpression
recipes
DNA >

DNA ISOLATION AND PLASMID PURIFICATION

> Phenol extraction and ethanol precipitation.

   

> Large Scale Plasmid Prep for 100 ML Superbroth Cultures

   

> Plasmid Miniscreen Protocol (Boil Prep)

   

> Alkaline Lysis Miniprep

   

> Alkaline Lysis Miniprep Modified 1

   

> Alkaline Lysis Miniprep Modified 2

   

> Amplification of plasmids with chloramphenicol

   

> Single-Strand DNA Prep. Large Scale

    > Titre of Bacteriophage
  > Large Scale Bacteriophage Prep

Modified ALKALINE LYSIS MINIPREP

Get a printable version of this protocol (requires Adobe Acrobat

This DNA can be used for transcription and translation in cell free assays, but since it contains no Rnase, when you map it with restriction endonucleases you will see a large smear of RNA. If your dropouts are larger than 500 bp, you will still be able to see them. However, if your dropouts are smaller it is a good idea to add some TE+RNase A to either your digests, or your loading buffer.  It differs from the standard protocol starting at step 7.

In the following protocol, plasmid DNA is prepared from small amounts of many different cultures of plasmid-containing bacteria. Bacteria are lysed by treatment with a solution containing sodium dodecyl sulfate (SDS) and NaOH (SDS denatures bacterial proteins, and NaOH denatures chromosomal and plasmid DNA). The mixture is neutralized with potassium acetate, causing the plasmid DNA to reanneal rapidly. Most of the chromosomal DNA and bacterial proteins precipitate -- as does the SDS, which forms a complex with potassium -- and are removed by centrifugation. The reannealed plasmid DNA is then concentrated by isopropanol precipitation and, RNA is removed by ammpmium acetate precipitation. The plasmid DNA is then concentrated by isopropanol precipitation. The plasmid DNA purified by this method can be used in in vitro transcription and translation.

Minipreps of Plasmid DNA

Most manipulations are performed in microcentrifuge tubes. This procedure is appropriate for preparation of small amounts of DNA from 1 to 24 cultures of plasmid-containing bacteria.

Materials
LB medium
Solution I (glucose-TE)
Solution II (NaOH/SDS solution)
Solution III (Potassium acetate solution)
100% ethanol
70% ethanol
Isopropanol
7.5M ammonium acetate
  1. Inoculate 5 ml LB medium with a single bacterial colony. Grow to saturation (overnight).
  2. Spin 1.5 ml of cells 20 sec in a microcentrifuge to pellet. Remove the supernatant with a Pasteur pipet, or aspirate using a pulled Pasteur pipet. This spin can be at 4C or at room temperature
  3. Resuspend pellet in 100 l glucose TE buffer (Solution I).(Let sit 5 min at room temperature;optional) Be sure cells are completely resuspended
  4. Add 200 l NaOH/SDS solution (Solution II), mix by tapping tube with finger,or inverting the tube gently twice,and place on ice.
  5. Add 150 l potassium acetate solution (Solution III) and vortex at highest speed for 2 sec to mix. Be sure mixing is complete
  6. Spin for 3 min in microcentrifuge to pellet cell debris and chromosomal DNA. This spin can be at 4C or at room temperature
  7. Carefully transfer the supernatant without any precipitate to another eppendorf and add 450uL 7.5M ammonium acetate. Mix by inverting tube and keep on ice 15 min. Centrifuge for 15 min. This step precipitates more than 90% of the high molecular weight RNA and a certain quantity of proteins, replacing organic extractions and RNase digestion.
  8. Transfer supernatant to another eppendorf and fill with isopropanol. Keep at -20 C for 10 min. and centrifuge for 10 min. Wash pellet with 70% EtOH, dry, and resuspend in 20uL TE. The plasmid DNA should be >90% supercoiled with no visible chromosomal DNA. Yield is approx. 10ug for Bluescript and pUC derivatives.

    This DNA can be used for transcription and translation in cell free assays, but since it contains no Rnase, when you map it with restriction endonucleases you will see a large smear of RNA. If your dropouts are larger than 500 bp, you will still be able to see them. However, if your dropouts are smaller it is a good idea to add some TE+RNase A to either your digests, or your loading buffer.

    Reference: A modification of Birnboim and Doly (1979) by Michael Bender via Yves (Shapiro lab)