DNA Sequencing Facility - Plasmid Preparation with PEG
This is a modified method by Kraft et al. (Kraft, R., J. Tardiff, K. S. Krauter, and L. A. Leinwand. 1989. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. BioTechniques 6:544-547). This protocol has been very effective for preparing plasmid DNA for sequencing but it is important to use FRESH PEG solutions during PEG precipitation. Some investigators often find it difficult to eliminate residual PEG, which is deleterious to sequencing reactions.
Reagents are listed below the protocol.
- Spin down a 3 to 4-ml overnight culture. Remove excess medium.
- Suspend the cell pellet in 100 ul TEG buffer containing 5 mg/ml lysozyme. Leave 5 minutes at room temperature.
- Add 200 ul fresh NaOH/SDS solution, mix well (do not vortex), and leave on ice 5 minutes.
- Add 150 ul KAc solution, mix well (do not vortex), and leave on ice.
- Centrifuge 10 minutes at 4C, pipet the cell lysate into a fresh tube containing 50 ul DNase-free RNase. Incubate for 30 minutes at 37C.
- Extract with phenol/chloroform. Precipitate with 1 ml cold 95% ethanol. Wash once with 70% ethanol. Dry.
- Dissolve the pellet in 100 ul proteinase K buffer. Add 1 ul proteinase K and incubate at 37C for 30 minutes.
- Extract with phenol/chloroform. Precipitate with 50 ul 7.5 M ammonium acetate and ethanol. Wash once with 70% ethanol. Dry.
- Dissolve the pellet in 15.2 ul water. Add 4.8 ul 5 M NaCl, mix, then add 20 ul 13% PEG. Mix well and leave on ice at least 1 hour.
- Centrifuge. Wash three times with 70% ethanol. Dry. Dissolve the pellet in 50 ul sterile distilled water. Electrophorese 1 ul on an agarose gel.
25 mM Tris-HCl, pH 8
50 mM disodium EDTA
LYSOZYME STOCK SOLUTION
20 mg/ml in water. Store at -20C
Prepare fresh from 10 N NaOH (40 g/100 ml) and 20% SDS. For 5 ml of solution, use 100 ul 10 N NaOH and 250 ul 20% SDS.
Combine 150 ml 5 M potassium acetate with 28.7 ml glacial acetic acid. Bring the volume to 250 ml with sterile distilled water. Filter sterilize and store at 4C.
DNase-FREE RNase A
20 mg/ml in water. Boil for 5 minutes and check for DNAase contamination.
PROTEINASE K BUFFER
10 mM Tris-HCl, pH 7.5
1 mM disodium EDTA
Autoclave, cool, then add 20% SDS to 0.5% final concentration.
13% in water. Store at room temperature.