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

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

Authors : 16.06.2010 - John Stewart

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

Contents 1 GROW UP 2 ALKALINE LYSIS 3 DNA PRECIPITATION and RESUSPENSION 4 REMOVE RNA, 5 POLYSACCARIDE and PROTEIN EXTRACTION 5.1 CTAB DNA Purification 5.2 Phenol / Chlorophorm Protein Removal 6 DNA PRECIPITATION and RESUSPENSION 7 PEG PRECIPITATION OF NON-SUPERCOILED DNA

[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.