PCR Primer Synthesis Tips
1. How are primers synthesized?
Currently, primer synthesis is mainly carried out using the solid-phase phosphoramidite triester method. This technique is widely used in DNA synthesizers, with major manufacturers like ABI/PE producing most of the equipment. Although different machines may vary in efficiency, reagent consumption, and cycle time, the basic synthesis mechanism remains the same. At Shenneng Gaming Company, we use the ABI3900 for high-throughput synthesis, while longer sequences are handled by Beckman 1000M and PE8909. For modified or high OD primers, we rely on the ABI394.
The phosphoramidite method involves immobilizing DNA on a solid support (CPG), where the synthesis proceeds from the 3' to the 5' end. Each nucleotide is added step-by-step through a 3'–5' phosphodiester bond. The process includes several key steps: deprotection, coupling, capping, and oxidation. After each cycle, the DMT protecting group is removed, allowing the next nucleotide to be added. This iterative process continues until the full sequence is completed.
Once synthesis is complete, the primers are cleaved from the CPG using ammonia, then purified using methods like OPC, PAGE, or HPLC. They are concentrated via C18, desalted, and precipitated before being suspended in water. Finally, the concentration is determined using an OD260 measurement, which helps quantify the yield accurately.
2. What are the methods of purifying the primers and how to choose them?
- C18 Desalting: This method removes salts but not small fragments. It's suitable for standard PCR applications but not for sequencing or cloning due to lower purity.
- OPC Purification: Based on the interaction between the DMT group and resin. Offers >95% purity and is ideal for primers up to 40mers.
- PAGE Purification: Uses denaturing polyacrylamide gel to isolate the desired fragment. Provides >95% purity and is best for long oligos (>50mers).
- HPLC Purification: High-performance liquid chromatography offers >99% purity, making it ideal for short or modified primers. However, it’s more expensive and less efficient for large-scale production.
3. How is the OD number of the primer quantified?
To determine the OD value, a UV spectrophotometer is used at 260 nm with a 1 cm quartz cuvette. The solution should be diluted to an absorbance between 0.2 and 1.0 for accurate readings. After dissolving the dried primer in a known volume of water, a 1 ml aliquot is measured. The OD value of the original stock is then calculated based on the dilution factor.
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