Protocol 1 Preparation Of Dna Templates For Sequencing

Purification of Double-Stranded Plasmid DNA by the Alkaline Method

Following denaturation, double-stranded plasmids containing DNA inserts of interest can be directly sequenced. One can simultaneously sequence both strands of the DNA insert at opposite directions using forward or reverse primers. The primers are annealed to the appropriate sites at their 3' ends of the DNA insert, in two separate sequencing reactions. This simultaneous sequencing approach can speed up the sequencing procedure and is especially useful for sequencing large DNA fragments. Double-stranded plasmids can be purified using the CsCl gradient method or the minipreparation method. Although we have found that the CsCl gradient method produces excellent results, it is time consuming and relatively expensive. The mini-preparation method, on the other hand, is simple, less expensive and gives excellent sequencing results. The detailed protocol for isolation and purification of plasmid DNA is described next.

1. Inoculate a single colony or 10 ml of previously frozen bacteria containing the plasmid DNA of interest in 5 ml of LB medium and 50 mg/ml of appropriate antibiotics (e.g., ampicillin), depending on the specific antibiotic-resistant gene carried by the specific plasmid. Culture the bacteria at 37°C overnight with shaking at 200 rpm.

2. Transfer the overnight culture into a microcentrifuge tubes (1.5 ml/tube) and centrifuge at 6000 rpm for 4 min. Carefully aspirate the supernatant.

Note: If the plasmid carried by bacteria is a high-copy number plasmid, 1.5 ml of cell culture usually gives a yield of approximately 40 mg plasmid DNA using the present method. In this way, 10 tubes of preparation can generate about 400 mg plasmid DNA, which is equivalent to a large-scale preparation. Therefore, the volume of bacterial culture used for purification of plasmid DNA depends on the particular experiment.

3. Add 0.1 ml of ice-cold lysis buffer to each tube and gently vortex for 1 min. Incubate at room temperature for 5 min.

Principle: The function of this step is to lyse the bacteria by hyperlytic osmosis, releasing DNA and other contents.

4. Add 0.2 ml of a freshly prepared alkaline solution to each tube and mix by inversion. NEVER VORTEX. Place the tubes on ice for 5 min.

Principle: This step denatures chromosomal DNA and proteins.

5. Add 0.15 ml of ice-cold potassium acetate solution to each tube. Mix by inversion for 10 s and incubate on ice for 5 min.

Principle: This step is to precipitate proteins, polysaccharides, and genomic DNA selectively. Under these particular buffer conditions, plasmid DNA still remains in the supernatant without being precipitated.

6. Centrifuge at 12,000 x g for 5 min and carefully transfer the supernatant to fresh tubes.

7. Add RNase (DNase-free, Boehringer Mannheim) to the supernatant at 1.5 ml/0.1 ml supernatant. Incubate the tubes at 37°C for 30 min.

Principle: RNase degrades total RNAs from the sample.

8. Remove RNase and other remaining proteins by using one of the following two options. Option A is faster, less expensive and more efficient than Option B.

Option A: Filtration Using a Probind Filter (Millipore)

Transfer the RNase-treated supernatant into a minifilter cup (0.4 ml/cup) placed in an Eppendorf tube. Cap the tube and centrifuge at 12,000 x g for 30 s. Remove the filter cup and the collected supernatant in the Eppendorf tube is ready for DNA precipitation. Proceed to step 9.

Note: The membrane filter attached at the bottom of the cup is specially designed to retain proteins and to allow nucleic acids to pass through. One cup can be reused for four to five times if one uses it for the same supernatant pool. However, it is not wise to reuse it for different plasmid samples due to potential contamination. The author routinely utilizes this type of filter for plasmid DNA preparations. DNA obtained is pure and of high yield with a ratio (A260/A280) of approximately 2.0.

Option B: Phenol/Chloroform Extraction a. Add an equal volume of TE-saturated phenol/chloroform to each tube and mix by vortexing for 1 min.

b. Centrifuge at 12,000 x g for 4 min and transfer the upper, aqueous phase into fresh tubes.

c. Add an equal volume of chloroform:isoamyl alcohol (24:1), mix by vortexing for 1 min and centrifuge as in the previous step.

d. Centrifuge at 12,000 x g for 4 min and transfer the supernatant to fresh tubes. Proceed to step 9.

9. Add 2 to 2.5 volumes of 100% ethanol to each tube. Mix by repeat inversions and allow the plasmid DNA to precipitate for 10 min at -80°C or 30 min at -20°C.

10. Centrifuge at 12,000 x g for 10 min. Carefully decant the supernatant, add 1 ml of prechilled 70% ethanol and centrifuge at 12,000 x g for 5 min. Carefully aspirate the ethanol and dry the plasmid DNA under vacuum for 15 min.

11. Dissolve the plasmid DNA in 15 ml of TE buffer or sterile deionized water. Take 1 ml to measure the concentration and store the sample at -20°C until use.

Reagents Needed

LB (Luria-Bertaini) Medium (per liter) 10 g Bacto-tryptone 5 g Bacto-yeast extract 5 g NaCl

Adjust pH to 7.5 with 2 N NaOH and autoclave. If needed, add antibiotics after the autoclaved solution has cooled to less than 50°C.

TE Buffer

Lysis Buffer

25 mM Tris-HCl, pH 8.0 10 mM EDTA 50 mM Glucose

Alkaline Solution 0.2 N NaOH 1% SDS

Potassium Acetate Solution (pH 4.8)

Prepare 60 ml of 5 M potassium acetate. Add 11.5 ml of glacial acetic acid and 28.5 ml of H2O. Total volume is 100 ml. This solution is 3 M with respect to potassium and 5 M with respect to acetate. Store on ice prior to use.

RNase (DNase-Free)

Boehringer Mannheim, Cat. No: 119915, 500 mg/ml.

TE-Saturated Phenol/Chloroform

Thaw crystals of phenol in 65°C water bath with occasional shaking. Mix equal parts of TE buffer and thawed phenol. Let it stand until the phases separate at room temperature. Mix an equal part of the lower phenol phase with an equal part of chloroform:isoamyl alcohol (24:1).

Chloroform:Isoamyl Alcohol (24:1) Ethanol: 100% and 70%

Purification of Single-Stranded DNA

Single-stranded DNA (ssDNA) utilized as a template usually gives excellent sequencing results. The following protocol works quite well for purification of ssDNA from plasmid-phage (phagemid) vectors, which include M13mp9, M13mp12, M13mp13, M13mp18 and M13mp19.

1. Streak an appropriate E. coli strain on an LB plate, which carries the putative phagemids containing the DNA insert of interest. Invert the plate and incubate at 37°C overnight in order to obtain individual colonies.

2. Inoculate a single colony in 3 ml of LB medium containing 50 mg/ml ampicillin and 12 mg/ml tetracycline or appropriate antibiotic. Incubate at 37°C overnight with shaking at 200 rpm.

3. Add 0.3 ml of the cell culture to 3 ml of superbroth in a 50-ml conical tube. Incubate at 37°C with shaking at 200 rpm for 2 to 3 h.

4. Add 8 ml of helper phage R408 (pfu = 1 x 1011, available from Stratagene) to the culture at step 3 and continue to culture for 8 to 10 h.

5. Centrifuge at 6000 rpm for 4 min and carefully transfer the supernatant to a fresh tube.

6. Based on the volume of the supernatant, add 1/4 volume of polyethylene glycol (PEG) precipitation buffer containing 3.5 M ammonium acetate buffer (pH 7.5) and 20% (w/v) PEG to the tube. Vortex for 1 min and leave it at room temperature for 20 min.

7. Centrifuge at 12,000 x g for 15 min and aspirate the supernatant completely.

8. Resuspend the PEG pellet in 1/4 volume (vs. the volume of the supernatant at step 5) of TE buffer (pH 8.0) and extract it with 1 volume of TE-saturated phenol:chloroform:isoamyl alcohol (25:24:1). Mix by vortexing and centrifuge at 11,000 x g for 5 min.

9. Carefully transfer the top, aqueous phase to a fresh tube and repeat extraction twice as in the previous step.

10. Measure the volume of the supernatant and precipitate the single-stranded DNA by adding 0.5 volume of 7.5 M ammonium acetate (pH 7.5) and 2.5 volumes of chilled 100% ethanol to the supernatant. Place at -70°C for 30 min.

11. Centrifuge at 12,000 x g for 20 min at 4°C and aspirate the supernatant. Wash the DNA pellet by adding 5 to 10 ml of 70% ethanol and spin down at 12,000 x g for 5 min. Carefully aspirate the ethanol and dry the pellet under vacuum for 20 min. Dissolve the DNA in 40 ml of TE buffer (pH 7.6). Take 2 ml to measure the concentration of single-stranded DNA at A260 and A280 nm. Store the sample at -20°C until use.

Reagents Needed

Superbroth Medium

Bacto-tryptone, 12 g Bacto-yeast extract, 24 g 0.4% (v/v) Glycerol

Dissolve in a total of 900 ml in dd.H2O and autoclave. Cool to about 50°C and add 100 ml of phosphate buffer containing 170 mM KH2PO4 and 720 mM K2HPO4. Autoclave again.

PEG Precipitation Buffer

3.5 M Ammonium acetate, pH 7.5

20% (w/v) Polyethylene glycol (PEG) 8000

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