Standard double deoxy DNA sequencing can be easily and efficiently detected using non-isobaric labeled chemiluminescence. In the sequencing reaction, biotin-labeled primers are used. Primers labeled with non-isotopic markers such as biotin derivatized primers can be used in a working scheme for the in-sequencing reaction set up for 5'-end labeled primers.
Operation method
Utilization of biotin-labeled primers
Materials and Instruments
DNA Move 1. Prepare a DNA touch plate mixture by mixing the following reagents in a microcentrifuge tube For more product details, please visit Aladdin Scientific website.
Biotin Tris-Cl MgCl2 DNA polymerase dATP dCTP dGTP dTTP
Thermocycler Nylon membrane Filter paper Centrifuge
(1) 1 μg of single-stranded DNA template or 1~3 μg of denatured double-stranded DNA template
(2) 0.5 pmol biotinylated DNA sequencing primer
(3) 2 μl 5×Bst reaction buffer
(4) Add water to 11 μl
(5) Heat to 70°C, then cool slowly to 30°C for more than 30 min.
2. While the template mixture is cooling, take 2 μl of each small portion of the A, C, T, G primer termination mixture and add it to the wells of 4 separate microcentrifuge tubes or microtitre plates, and then make the markings of A, C, G, T.
3. Preheat the tubes or titration plates containing the termination mixture at 65 °C.
4. Add 1 μl of Bst DNA polymerase to the cooled template mixture from step 1.5. Add 2.5 μl of polymerase/template mix to each primer termination mixture prepared in step 2 and incubate at 65°C for 5 min.6. Dilute the 20×addition solution reservoir with water to 1×addition solution, add 2 μl of 1×addition solution to each sample tube or well, and incubate at 65°C for 5 min.
7. Add 4 μl of termination solution to each tube or well.8. Prepare standard 0.2-0.4 mm thick sequencing gel containing 8 mol/l urea.
9. Before adding samples, the samples were warmed at 80°C for 2 min, placed in an ice bath, 2~3 μl of samples were added, electrophoresed, and the migration of the tracer dye was monitored.10. Cut three pieces of Whatman 3MM filter paper to the size of the gel or slightly larger, and cut a piece of nylon film to cover the area of the gel to be transferred.11. Disassemble the gel unit and separate the glass plates.
12. Place a piece of Whatman 3MM dry filter paper over the gel and carefully lift the gel by peeling up the filter paper.
13. Place the filter paper with the gel attached, gel side up, on the glass plate.
14. Submerge the nylon membrane in TBE buffer or spray TBE buffer with a squirt bottle to wet it completely.
15. Carefully place the moistened membrane on the gel and roll the membrane over with a pipette or smooth glass rod to evacuate air bubbles.
16. Place two sheets of Whatman 3MM dry filter paper on top of the membrane, place another glass plate on top of the filter paper and place a 2-4 kg weight on it and transfer for 1 h. The membrane is then transferred to the gel.
17. Separate the sequencing gel/membrane sandwich by carefully peeling the membrane from the gel and marking the side of the membrane that is in contact with the gel with a pencil, place the membrane on filter paper and allow to dry for approximately 10 min.
18. Using a UV crosslinking device, UV transilluminator or hand-held UV lamp, irradiate the transfer membrane to immobilize the DNA with a total UV irradiation of 120 MJ/cm2.
19. Proceed to step 20 or place the membrane between two layers of plastic wrap and store at 4 °C for up to 6 months.20. Place the membrane in a large, heat-sealable bag, add 500 ml of Blocking Solution I, carefully remove air bubbles, seal the bag, and leave for 10 min.
21. Dispose of the membrane as follows
(1) 200 ml of coupling buffer for 20 min
(2) 500 ml blocking solution 15 min
(3) 500 ml wash buffer Ⅰ 10 min, 3 times
(4) 500 ml analysis buffer 2 min
(5) 50 ml dioxane assay 5 min
21. Drain excess test solution from the bag, flatten any wrinkles, and reseal the pocket.
22. Expose the wrapped film by placing it in direct contact with the X-ray film at room temperature.