Experiments on the preparation of denaturing templates for sequencing by double deoxy chain termination method
Experiments on the preparation of denaturing templates for sequencing by double deoxy chain termination method
The following method of denaturing plasmid DNA by base denaturation should be used in conjunction with Scheme 3, as sequencing enzymes are used to catalyze the double deoxy sequencing reaction in this process. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experiments on the preparation of denaturing templates for sequencing by double deoxy chain termination method
Materials and Instruments
Ammonium acetate Chloroform Isoamyl alcohol DMSO Ethanol NaOH Phenol Sequencing reaction buffer Agarose gel Oligonucleotide primers Closed loop double stranded plasmid DNA Move makings For more product details, please visit Aladdin Scientific website.
Dry ice-ethanol bath 65°C water bath
Buffers and solutions
See Appendix 1 for the composition of reagents, buffers, and reservoirs, and dilute the reservoirs to the appropriate concentration before use.
Ammonium acetate (5 mol/L, unbuffered, pH~7.4)
Chloroform: isoamyl alcohol (24:1, V/V)
DMSO
Optional, see step 10.
Ethanol
NaOH(2mol/L)/EDTA(2 mmol/L)
Dilute 10mol/L sodium hydroxide solution to the appropriate concentration before use.
For phenol, equilibrate with water or TE (pH 7.6).
Sequencing Reaction Buffer
Gel
Agarose gel (0.8% m/V)
Nucleic Acids and Oligosynucleotides
Oligonucleotide primers
Oligonucleotide primers used for denatured double-stranded DNA template sequencing are generally 20-30 nucleotides longer than those used for single-stranded DNA template sequencing.
The use of longer primers for denaturing double-stranded DNA template sequencing can reduce the occurrence of false bands.
For a list of common primers that incorporate vector sequences upstream of the target region, see "Common Primers" in the Information Section of Chapter 8, and refer to "DNA Sequencing Primer Reservoir Preparation" in the Information Section of this chapter.
Closed-loop double-stranded plasmid DNA
Small- and large-scale preparations of plasmid DNA from cultured organisms are described in Chapter 1, and can also be performed according to the alternative methods described later in this protocol.
A set of four sequencing reactions (ddA,ddG, ddC, ddT) requires 5ug of DNA.
Specialized Equipment
Dry ice-ethanol bath
65°C water bath
Methods
1. 5ug of purified plasmid DNA was added to a 1.5ml microcentrifuge tube and refilled with water to a total volume of 50ul. 10ul of freshly prepared 2mol/LNaOH/2 mmol/LEDTA solution was added and allowed to stand at room temperature for 5 min.
2. Add 30ul of 5mol/L ammonium acetate and mix by vortexing and shaking.
3. Add 45ul of equilibrium phenol and mix with vortexing and shaking.
4. Add 135 chloroform/isoamyl alcohol and mix by vortexing and shaking. Then centrifuge at room temperature for 15 min at maximum speed in a microcentrifuge.
5. Carefully pipette the upper aqueous phase into a clean microcentrifuge tube, add 330ul of ice pre-cooled ethanol and mix well. Place in a dry ice-ethanol bath for 15 min.
6. Recover denatured DNA by centrifugation in a microcentrifuge for 15 min at maximum speed.
7. Pour off the supernatant carefully without stirring the DNA precipitate, which may or may not be visible on the wall of the tube.
8. Re-centrifuge for 2S and use a tip to aspirate any remaining trace ethanol without stirring the DNA precipitate.
9. Cool and dry the DNA precipitate at room temperature, then re-dissolve the DNA in distilled water to a final concentration of lug/ul.
10. Anneal the primer DNA to bind to the denatured template, using 5ug (5ul) of base-denatured plasmid DNA for a set of four DNA sequencing reactions (e.g., 1.25ug of denatured DNA for reactions containing ddA; 1.25ug of denatured DNA for reactions containing ddG; and so on). Set up the annealing reaction as follows:
Base denatured DNA 5.0ug (dissolved in 5ul of water)
DMSO ~undefined optional) 2.0ul
Sequencing reaction buffer 2.0ul
Sequencing primer 10.ul(10ug)
Water to final volume 11.0ul
~See Solution 4 for troubleshooting.
Heat the mixture to 65°C for 2 min, then allow to cool slowly to room temperature. Keep the annealed sample on ice until the extension/chain termination reaction is performed.
11. Proceed as in Scheme 3, step 3. 