Primer synthesizer is the core equipment of molecular biology laboratory, and its efficient and stable operation is crucial for experiments. The three most common types of faults are pipeline blockage, valve leakage, and abnormal synthetic data. This article will provide a brief analysis and offer targeted solutions.
1、 Pipeline blockage
This is a common malfunction that directly leads to synthesis failure.
Diagnosis: Usually accompanied by abnormal increase in column pressure, poor reagent delivery, and extremely low or no final product.
Main reasons and solutions:
Composite column blockage: the most common occurrence. Caused by precipitation of monomers or base solutions, drying of column beds, or solid particles. Solution: Immediately interrupt the synthesis and use strong anti blocking solvents (such as acetonitrile, concentrated TCA/dichloromethane solution) to reverse or forward flush the synthesis column and upstream pipelines. It is necessary to ensure that the reagents are fresh, anhydrous, and thoroughly rinsed after synthesis in daily life.
Micro blockage of pipelines or valves: High pressure in specific reagent pipelines. Caused by crystallization or degradation products. Solution: Implement a "Purge" cleaning program for specific reagents, or manually disassemble and clean the relevant valve blocks and capillary tubes.
2、 Valve and pipeline leakage
Leakage can lead to inaccurate reagent measurement, cross contamination, and even damage to equipment.
Diagnosis: The pressure cannot be established or continues to decrease, there is liquid accumulation around the reaction column, the synthesis yield is low, and there are "missing" or erroneous additions in the sequence.
Main reasons and solutions:
Aging or wear of sealing rings: The rotor sealing gasket or pipeline joint sealing ring of valves (especially main distribution valves) is the most common leakage point. Solution: Regularly replace all critical sealing rings according to the maintenance plan. After discovering a leak, immediately shut down the machine and replace the corresponding sealing components according to the manual.
Loose or broken pipeline connections: Check if the fittings of each card sleeve are tightened and inspect for creases or cracks in the PTFE capillary tube. Solution: Re tighten or replace damaged pipelines.
3、 Abnormal synthesis data
Manifesting as low yield and poor purity (especially with the failure of long-chain primers), but the instrument has no alarm.
Diagnosis: HPLC/mass spectrometry analysis showed incorrect product, multiple truncated sequences, or a much lower than expected full-length yield.
Main reasons and solutions:
Reagent issues (most common): degradation of monomer solutions (especially G monomers), failure of activators or oxidants, and excessive water content in acetonitrile. Solution: Strictly manage reagents, use fresh, high-purity, anhydrous reagents and acetonitrile, and regularly replace desiccants.
Low efficiency of de capping/de protection: Insufficient concentration of de capping solution or final cutting/de protection reagent (such as concentrated ammonia water) or incorrect time setting. Solution: Ensure accurate reagent preparation and verify the adequacy of the deprotection reaction procedure.
Software or parameter errors: Although rare, incorrect synthesis sequences or loop parameters can lead to systematic failures. Solution: Carefully check the sequence file and synthesis parameter settings before each synthesis.
Summary: Prevention is better than cure. Establishing a regular preventive maintenance plan (such as monthly cleaning, quarterly replacement of sealing rings, monitoring of reagent expiration dates) and maintaining detailed work logs is an effective strategy to minimize failures and ensure the success rate and quality of primer synthesis. When encountering complex problems, equipment engineers should be contacted in a timely manner.