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Midi-Prep Plasmid Purification - Alkaline Lysis

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Extraction and Purification


Branch: Sci-Wiki
Type: Idea
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
16.06.2010 - John Stewart   


A midi-prep of high copy number plasmid (circa. 4kb) should yield around 50-400 micrograms (ug) of plasmid.

Contents

[edit] GROW UP

  1. 50 ml Superbroth + 50ul Ampicillin (100 mg/ml) + Bacteria containing plasmid; O/N at 370C in shaker.
  2. Transfer to Falcon tubes and spin: 10' at 4K rpm.

[edit] ALKALINE LYSIS

  1. Drain well; resuspend pellet thoroughly in 6ml of Solution 1.
  2. Tip in one motion 12ml Solution 2 from Falcon tube in to suspended bacteria; immediately invert 3 times; place on ice until solution clears; typically 5'-10' on ICE (not more than 10').
  3. Dump 8ml Solution 3 (KOAc) from Falcon tube; invert gently at first, and then every 5'; keeping solution for at least 20' on ICE.
  4. Centrifuge 10' at 4K; pour s/n through 2x Kimwipes (or filter paper) to remove white precipitate; and collect clear solution in clean tube.

[edit] DNA PRECIPITATION and RESUSPENSION

  1. Add 0.6 vol isopropanol; mix well; 20' at RT.
  2. Centrifuge 10'-20' at 4K.
  3. Discard s/n; wash with 70% Ethanol; allow to dry; and resusp in 500ul TE dislodging pellet (allow white or clear pellet to completely disolve (5'-60')).
  4. Transfer to eppendorf; centrifuge 15' at 13K to remove additional precipitate; and transfer s/n to new eppendorf.

[edit] REMOVE RNA,

  1. Add 2ul RNase (10mg/ml); and leave for 20' at 370C.

[edit] POLYSACCARIDE and PROTEIN EXTRACTION

This step may be achieved with CTAB or Phenol/Chlorophorm extraction, both are effective, CTAB is better at removing polysaccarides, while Phenol/Cholorophorm might be better at removing protein and leaving more DNA.

Either:

[edit] CTAB DNA Purification

  1. Add at least 1/5th vol 5M NaCl; mix thoroughly;
  2. Add 1/8th vol CTAB; mix thoroughly; and leave for 10’ at 650C.
  3. Add equal volumn of Chloroform-amyl alchohol; mix thoroughly; and spin for at least 10’ at 4K.
  4. Take top layer; and if necessary repeat step 3. until no white layer occurs at interface.

OR,

[edit] Phenol / Chlorophorm Protein Removal

  1. Add 1/10th volumn of 3M NaAc pH8.9 + equal vol of phenol/chlorophorm; vortex; and centrifuge for 10' at 4K.
  2. Remove aqueous top layer to fresh tube; and repeat phenol extraction (not salt) until no white precipitate occurs at interface (at least 2, up to 6 times).
  3. If more than 2 phenol extractions are required, repeat a further time using chloroform instead of phenol to remove residual phenol.

[edit] DNA PRECIPITATION and RESUSPENSION

  1. Add equal volumn isopropanol (400-450ul); mix well; and leave for at least 15' at RT.
  2. Centrifuge for 15' at 13K; wash pellet with 70% Ethanol; and allow to dry.
  3. Resuspend pellet in 500ul TE (try to dislodge the pellet from the bottom of the tube; it may be white or clear and may take minutes or hours to resuspend- clear pellets can be seen by gently inverting the solution against pale light).

[edit] PEG PRECIPITATION OF NON-SUPERCOILED DNA

Optional, but recommended for DNA used in transfections or injections.

  1. Add 500ul 13% PEG-8000 in 1.6M NaCl; at least 1hr RT (better final yields if left O/N in cold room).
  2. Centrifuge 15' at 13K; wash pellet in 70% ethanol; and allow to dry thoroughly.
  3. Resuspend in 100-200ul TE pH8.




  • This page was last modified 19:51, 16 June 2010.
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