Sequencing reaction experiments

Summary

To get the right signal intensity, adjust the amount of DNA according to the size of the DNA fragments. For ABI PRISM Big Dye Terminator Ready Reaction mix (Applied Biosystems) and GeneAmp PCR System9700 (Applied Biosystems) thermal cyclers. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Sequencing reaction experiments

Materials and Instruments

Cycle Sequencing Kits Primers and Templates
Thin Wall PCR Tubes Thermocyclers GeneAmpPCRSystem9700 Instrument

Move

I. Materials

1. Buffers and solutions

ABI PRISM Big Dye Terminator Ready Reaction mix (Applied Biosystems) or other cycle sequencing kits

2. nucleic acids and oligonucleotides

Primers and templates

3. Specialized equipment

Thin-walled PCR tubes for DNA amplification

Thermal cycler, GeneAmp PCR System 9700 (Applied Biosystems) instrument or equivalent

Methods

1. Add the appropriate amount of primer and template (as described above) to the PCR tube.

2. Add 4ul of Terminator Ready Reaction mix to each tube.

This is half the amount recommended in the instructions, which is sufficient and saves reagents.

3. Make up to 20ul with deionized water, mix well, and centrifuge briefly.

4. On a GeneAmp PCR System 9700 (Applied Biosystems) or equivalent, perform the following procedure.


5. After the previous step, the reaction product can be purified immediately or stored at -20°C for future purification.

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