• GTE buffer ( 50 mM glucose, 25 mm Tris-HCl(pH 8.0),
• 10 mm EDTA (pH 8.0), (200 µL/tube)
• 0.2N NaOH / 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 NaCl
• 13% PEG 8000 (sterilized by autoclaving, rather than by filtration)
• Deionized H2O * Terrific Broth (1 L)

To prepare Terrific Broth, add 100mL of a sterile solution of 0.17 M KH2HPO4 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 = 5.0g.

Procedure

1. Incubate cultures overnight at 37ƒ C 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 of 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 pipeting 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. NOTE: Throughout this procedure, the use of a vortex must be avoided so as to minimize shearing of the contaminating chromosomal DNA.
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 be 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 37ƒ C 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.
   NOTE: Inadequate extraction will result in poor sequencing data. An additional CHCl3 extraction may be necessary if debris is still visible at the interface after the second extraction.
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 uL of deionized H2O. and precipitate the plasmid DNA by first adding 8.0 µL of 4M 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 ƒC in a fixed-angle rotor. NOTE: The temperature parameter is very important; adhere to the recommended 4ƒC NOTE: If you use a horizontal rotor, do not aspirate the supernatant in Step13 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 non-horizontal rotor. Carefully remove the supernatant and rinse the pellet with 500µL of 70% ethanol. Then dry the pellet under vacuum for 3 min, resuspend in 20 µL of deionized H2O, and store at -20ƒC.

 

Appendix B: Extension Primer Recommendations

Sequence Selection, Synthesis, and Purification
Your decisions concerning primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data obtained in Taq DyeDeoxy terminator cycle sequencing reactions. These decisions are particularly important when sequencing is done on real-time detection systems where signal strength is critical. The purpose here is to provide you with guidelines to follow in making these important decisions. Some of theses recommendations are based on information that is general knowledge amongst researchers, while others are based on our experience with DyeDeoxy terminators.
Recommendations

• Primers with long runs of a single base (i.e., more than three or four, especially G or C) should be avoided.
• Primers should generally be at least 18 bases long to ensure good hybridization.
• For cycle sequencing, primers with melting temperature above 45ƒC generally produce better results than primers with lower melting temperatures.
• For primers with a GC content of less than 50%, it may be necessary to extend the recommended lower limits of 45ƒC.
• Use of primers longer than 18 bases also minimizes the chances of encountering problems with a secondary hybridization site on the vector or insert.
• Primers that have secondary structure, or that can hybridize to form dimers, should be avoided.
• Polystyrene 40-nanomole columns are the best choice for primer synthesis. These columns afford the highest-quality primer syntheses with yields of purified oligonucleotide sufficient for many sequencing reactions.
  

The facility has available for use the computer program Oligo 5.0, which has been helpful for primer selection for several investigators at the University.