A key determining factor underlying the success or failure of Taq DyeDeoxy terminator cycle sequencing reactions is the quality of the template DNA. This is particularly important when sequencing is done on real-time detection systems where signal strength is critical. Perkin-Elmer has improved a common template preparation procedure so that it can now be used to reliably provide ample quantities of sequenceable template DNA for such reactions. The newly modified method is a mini alkaline-lyses/PEG precipitation procedure that is inexpensive and relatively easy to perform on a large number of samples in most laboratories. This procedure provides an attractive alternative to more expensive and/or time-consuming methods currently used to prepare cosmid or plasmid DNA for Taq cycle sequencing reactions using DyeDeoxy terminators.

Advantages of the Improved Procedure

The modified mini alkaline-lysis/PEG precipitation procedure can be used to isolate template DNA from 12 to 24 plasmid samples in approximately three hours, with yields of 5-30 µg of DNA per 1.5 mL of culture, depending on the host strain and the plasmid vector. The method has proven to be reproducible and consistently yields template DNA suitable for use with the PRISM Ready Reaction Kit protocol. One of the key features of the improved procedure is the growth of bacteria in Terrific Broth (1), instead of Luria Broth. This results in a four-to eight-fold increase in the number of bacteria per milliliter of medium, which leads, in turn, to higher plasmid yields.

Another important factor influencing maximum plasmid yields is the manner in which the bacteria are propagated. Adequate aeration must be provided; otherwise, the bacteria will undergo anaerobic growth that will result in significantly reduced numbers of bacteria per milliliter of medium. By providing proper aeration during bacterial growth, enough DNA to complete an entire gene walking project (based on a 3 kb gene and 50 µg of template DNA) can be purified routinely from one or two plasmid preparations.

Another key feature of the improved protocol is the inclusion of PEG precipitation step. This precipitation yields high-quality, super-coiled plasmid DNA that is relatively free of contaminating chromosomal DNA and RNA. In fact, plasmid DNAs that have been isolated using other protocols, and that could not be sequenced with Taq DyeDeoxy terminators, have been cleaned up by a PEG precipitation step and then sequenced successfully using the Taq DyeDeoxy terminator chemistry.

Host Strain Recommendations

The quality of the template DNA prepared by even the best methods can be significantly compromised depending on the bacterial strain from which the DNA was isolated. We have evaluated many commonly used host strains for their impact on the sequenceability of several ds templates isolated by the recommended template preparation methods. Based on these evaluations, we make the following recommendations for host strains.

Highly Recommended	Recommended	Not Recommended
DH5a	JM109	JM101
HB101	XL1Blue
	MV1190

The difference between highly recommended and recommended is that on a limited number of occasions (<10%), ds templates isolated from the recommended host strains would yield less than acceptable sequencing results when compared with sequencing results obtained from the same ds templates isolated from the highly recommended host strains. Not recommended means that, at least 50% of the time, the DNA isolated from this strain would not yield acceptable sequencing results.

The Modified Alkaline-Lysis/PEG Precipitation Procedure

Materials

• GTE Buffer (50 mM glucose, 25 mM Tris-HC1 (pH 8.0), 10 mM EDTA (pH 8.0), 200 µL/tube)

• 0.2 N Na0H / 1% SDS (freshly made) (300 µL/tube)

• 3.0 M Potassium acetate (pH 4.8) (300 µL/tube)

• RNase A (DNase-free) (10 mg/mL) (2 µL/tube)

• Chloroform (400 µL/tube)

• Isopropanol, 100%

• Ethanol, 70%

• 4.0 M NaC1

• 13% PEG₈₀₀₀ (sterilized by autoclaving, rather than by filtration)

• Deionized H₂0

• Terrific Broth (1 L)

To prepare Terrific Broth, add 100 mL of a sterile solution of 0.17 M KH₂PO₄ and 0.72 M K₂HPO₄ to 900 mL of base broth (base broth = 12g bacto-tryptone, 24g bacto-yeast extract, 4.0 mL glycerol, q.s. to 900 mL with deionized H₂O and then autoclave).

Note:

The above volume of glycerol is most easily measured out by weighing: 4.0 mL glycerol = 5.0 g.

Methods

1. Incubate cultures overnight at 37 ºC in Terrific Broth, with an appropriate amount of antibiotic, in Erlenmeyer flasks or 50 mL polypropylene tubes. (To maintain adequate aeration in the flasks or tubes, restrict the culture volume to be no more than one quarter of the total flask volume, or one fifth of the total tube volume.)

2. Pellet 1.5 mL aliquots of culture for 1 min in a microcentrifuge.

Note:

A total culture volume of 4.5 mL can be spun down per tube without changing volumes in the procedure. This allows you to achieve a three-fold increase in yield while eliminating the need for extra tubes and additional handling.

3. Remove the supernatant by aspiration and resuspend the bacterial pellet in 200 µL of GTE buffer by pipetting up and down.

4. Add 300 µL of freshly prepared 0.2 N NaOH / 1% SDS and then mix the contents of the tube by inversion until the solution clears. Then incubate on ice for 5 min.

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   Note:

   Throughout this procedure, the use of a vortex must be avoided so as to minimize shearing of the contaminating chromosomal DNA.

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   5. Neutralize the solution by adding 300 µL of 3.0 M potassium acetate, pH 4.8, mix by inverting the tube, and incubate on ice for 5 min.

   6. Remove cellular debris by centrifuging for 10 min at room temperature, and then transfer the supernatant to a clean tube.

   7. Add RNase A (DNase-free) to a final concentration of 20 µg/mL and incubate the tube at 37 ºC for 20 min.

   8. After the RNase A treatment, extract the supernatant twice with 400 µL of chloroform. Mix the layers by hand for 30 s after each extraction. Centrifuge the tube for 1 min to separate the phases and remove the aqueous phase to a clean tube.

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   Note:

   Inadequate extraction will result in poor sequencing data. An additional CHCI3 extraction may be necessary if debris is still visible at the interface after the second extraction.

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   9. Precipitate the total DNA by adding an equal volume of 100% isopropanol and immediately centrifuging the tube for 10 min at room temperature.

   10. Wash the DNA pellet with 500 µL of 70% ethanol and then dry under vacuum for 3 min.

   11. Dissolve the pellet in 32 µL of deionized H₂O, and precipitate the plasmid DNA by first adding 8.0 µL of 4 M NaCl, and then adding 40 µL of autoclaved 13% PEG₈₀₀₀.

   12. After thorough mixing, incubate the sample on ice for 20 min, and then pellet the plasmid DNA by centrifugation for 15 min at 4 ºC in a fixed-angle rotor.

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   Note 1:

   The temperature parameter here is very important; adhere to the recommended 4 ºC.

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   Note 2:

   If you use a horizontal rotor, do not aspirate the supernatant in Step 13 because the clear pellet adheres to the bottom of the tube and can be lost if you are not careful. Remove the supernatant by decanting. Either approach, decanting or aspirating, can be used to remove the supernatant from a tube spun in a nonhorizontal rotor.

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   13. Carefully remove the supernatant and rinse the pellet with 500 µL of 70% ethanol. Then dry the pellet under vacuum for 3 min, resuspend in 20 µL of deionized H₂O, and store at -20 ºC.

   
   References

   1. Tartof, K.D. and Hobbs, C.A., "Improved Media for Growing Plasmid and Cosmid Clones, "Bethesda Res. Lab. Focus 9:12 (1987).
   Back to DNA Sequencing Protocols

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