Cycle sequencing: double deoxy sequencing experiments using PCR and primer end-labeling
Cycle sequencing: double deoxy sequencing experiments using PCR and primer end-labeling
The key to the success of this method is the purity of the template DNA. Contamination with agarose, salt, and protein can lead to premature termination or suspension of the DNA polymerase, while oligonucleotides from DNA and RNA degradation will cause primer mismatches. These contaminations can seriously contribute to the appearance of false positive bands or blanks. 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
Cycle sequencing: double deoxy sequencing experiments using PCR and primer end-labeling
Materials and Instruments
ddNTP Extension Termination Mixture Enzymes and Buffers AmpliTaq CS or other exonuclease-free DNA polymerase Cyclic Sequencing Buffer Heat-stabilized DNA polymerase Nucleic acids and oligonucleotides Template DNA Move makings For more product details, please visit Aladdin Scientific website.
Small centrifuge tubes or microplates Thermocyclers
Solutions and buffers
Refer to Appendix 1 for formulations of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
ddNTP Extension/Termination Mixture
ddNTP solution: four ddNTPs, 5.0 mmol/L each
dNTP solution: four dNTPs, 1.0 mmol/L each
EDTA (0.05mol/L, pH 8.0)
Formamide Sampling Buffer
Tris-Cl(1mol/L,pH8.0)
Enzyme and its buffer
AmpliTaq CS or other DNA polymerase without exonuclease activity (5units/ul).
5X Cycle Sequencing Buffer
200 mmol/L Tris-Cl (pH 8.8)
25 mmol/LMgCl2
Heat-stable DNA polymerase
When selecting an enzyme, refer to the introduction to this chapter and to Table 12-19 in Method 5 to select a sequencing system. The method is based on AmpliTaqCS. However, other similar heat-stabilized enzymes are also available.
Nucleic Acids and Oligonucleotides
Oligonucleotide primers, 5' end radiolabeled with 33P or 32P
See Chapter 10, Scheme 2.
Template DNA
Plasmids, stickies, λ phage and phage M13DNA, as well as DNA obtained by any of the methods in Chapters 1-4, can be used as templates. Table 12-12 shows the amount of each template required (also refer to Table 12-4).
Specialty Devices
Small centrifuge tubes (0.5 ml) or microtiter wrenches (soft, heat-stable, 96 300ul solvent U-wells)
These plates can be labeled in different colors and/or marked with C, T, A and G.
Designed thermal cycling program (see step 5).
Methods
1. Place 4 ul of each ddNTP mixture (refer to Table 12-11) into the appropriate 0.5 ml centrifuge tube or microtiter plate. Place on ice. 
2. add to each tube/well:
Double-stranded DNA template 10~100fm
1.5pmoles primer radiolabeled at the 5' end by 32P 1.0ul
5X cyclic sequencing buffer 2.0ul
Add water to 5.0ul
~undefined if using primers labeled at the 33P end: use 5pm standard preparation of oligonucleotides radiolabeled by 33P (see Appendix Methods: Cyclic Sequencing Reactions Using PCR and [ a-32P ]dNTP Internal Labeling to ensure stoichiometry of the sequencing mixture. The amount of DNA can be increased 3-fold (30-300 fm) without increasing the reaction volume.
3. dissolve AmpliTaq CS polymerase to 0.5-1.0 U/ul in 1X Cycle Sequencing Buffer. add 1ul of the dissolved enzyme solution to the well or tube.
4. Flick the tube wall or shake the microplate to mix the substances. If necessary, add a drop of mineral oil to the reaction, cap the tube, and centrifuge at 2000 r/m for 2 s. Make sure the mineral oil has not been exposed to UV light. Otherwise it will produce a strong inhibitor of PCR.
5. Place the tube or microplate in the PCR machine and preheat to 95°C. Follow the procedure below.
IMPORTANT: Do not delay in starting the program. Ensure that the preheating time at 95°C is not more than 3 min, otherwise the DNA polymerase will be inactivated. 
6. Remove the plate or tube and add 5ul of formamide spiking buffer to each plate or tube.
7. The reaction can be stored at -20°C for 5 days or analyzed directly by denaturing polyacrylamide gel electrophoresis (Scheme 8,9,10,11 and 12). After denaturation (100°C for 2 min), quench on ice and sample with 3 ul of each reactant. 
