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Maxi 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: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
18.06.2010 - John Stewart   


A maxi-prep of high copy number plasmid (circa. 4kb) should yield around 0.1-10 milligrams (mg) of plasmid.

Contents

[edit] GROW UP

  1. Add 50ml SuperBroth + 100ul Ampicillin (100 mg/ml) + Bacteria; and shake for at least 3 hours at 370C (or o/n).
  2. Tip cultured bacteria into 1 or 2 flasks containing 600ml Superbroth + 600ul Ampicillin (100 mg/ml); and shake overnight at 370C.

[edit] ALKALINE LYSIS

  1. Transfer to tubes; and centrifuge for 10' at 5K (based on Sorval centrifuge);
  2. Discard S/N; fully resus each pellet in 5ml of Solution 1; before adding Solution 1 to final volumn of 80-160ml.
  3. Dump 150-300ml Solution 2 in one smooth action; and leave for 5'-10' on ICE.
  4. Dump 100-200ml cold Solution 3; and leave for at least 20' on ICE.
  5. Centrifuge for 15' at 9K; and then pour S/N through double layer of filter paper or Kimwipes.
  6. Centrifuge again for up to 30’ at 13K (optional, but it helps remove protein and other precipitates).

[edit] PRECIPITATION OF DNA (1)

  1. Add 0.6 vol isopropanol to S/N; 20' at RT.
  2. Centrifuge 30' at 4K or 15' at 6K; and discard S/N.
  3. Wash pellet in 70% ethanol; allow to dry; and then resuspend in 5-10ml (total) TE pH8.
  4. Centrifuge for at least 15’ at 13K (to remove excess protein); and keep S/N.

[edit] BREAKDOWN OF RNA

  1. Add 10ul (total) RNase (10mg/ml); and leave for 30' at 37C.
  2. Note:

[edit] REMOVAL OF NON-SUPERCOILED DNA by PEG PRECIPTATION

TIP: phenol extractions may be performed before or after PEG precipitation. Isopropanol precipitation of the DNA must preceed PEG preciptitation in either case. If you are gentle with your DNA during Phenol / Cholorophorm extraction, it should not matter.

  1. Add equal vol 13% PEG-8000 in 1.6M NaCl; and leave over night in cold room (lower yields can be expected if left for at least 60' on ICE).
  2. Centrifuge for 30' at 4K or 10' at 10K; and discard S/N.
  3. Wash pellet with 70% ethanol; and allow to dry.
  4. Resuspend pellet in 5ml TE pH8 (may take hrs); and transfer to single tube.

[edit] PHENOL/CHOLOROFORM EXTRACTION

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

[edit] ISOPROPANOL PRECIPITATION OF DNA (2)

  1. Add 0.6 vol isopropanol; mix well; leave for at least 20' at RT or longer on ice; and centrifuge for 10' at 4K.
  2. Discard S/N; wash pellet in 70% Ethanol; allow to dry; and resus pellet in 10ml of PBS (phosphate buffer saline) (pref O/N rocker).

[edit] TRITON X EXTRACTION- ENDOTOXIN REMOVAL

This is an optional step, which removes bacterial contaminants that can effect DNA immunizations (for example).

  1. Add 0.1ml of Triton X-114; mix well using transfer pipette (should be cloudy); and leave for 5' on ICE (until solution clears).
  2. Heat to 450C until solution becomes cloudy (takes approx 5' at 450C).
  3. Centrifuge 5' at 4K at RT.
  4. Keep top (aqueous) layer; add PBS to 10ml; and repeat Triton extraction X 3 times.
  5. Transfer final S/N to falcon tube.

[edit] ISOPROPANOL PRECIPITATION OF DNA (3)

  1. Add 1ml 3M NaAc pH5 (mix) + 10ml isopropanol; mix well; and leave O/N in cold room (shorter durations on ice will yield less DNA).
  2. Centrifuge for 10' at 4K; discard S/N; wash clear (sometimes milky at first) pellet in 70% ethanol; allow pellet to dry; and resuspend in 1- 2 ml of normal saline (for injections), PBS or TE (for storage).




  • This page was last modified 18:33, 18 June 2010.
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