We have a certain protocol that we follow in lab for a PCR test that we do with our DNA. We have been doing this same protocol for the past 2 years. For the first year, it has worked just fine. Recently, however, we have begun to see what looks like contamination even in our water. No matter how often the water is replaced or anything, the band will often appear. We are using the same conditions as we have been for the past 2 years. Has anyone ever noticed a decline in the performance of a PCR machine, or differences when they run PCR machine versus another?
It is certainly a good idea to calibrate PCR machines time to time, and for sure there can be differences between machines, or even between wells. However, when it comes to contamination it is always the best option to throw all reagents away, UV/EtOH clean everything and re-order or use a different batch of reagents.
Péter Gyarmati thanks for that. Yes, we have thrown out reagents, bought a completely new kit, clean the room with ETOH and nuclease spray, ordered new primers, and still get this contamination. I have exhausted all options of why this band keeps appearing.
Yes, I have. Some older machines do not change temperature as fast and so may need the steps to be longer. Other issues may be calibration, cleaning, and even how steady the electricity is coming into the facility. Sometimes a step-down clean up adaptor is needed if the voltage is unstable.
Contamination is unlikely to be related to your thermocycler. Reagents are more likely an issue. Did you autoclave or otherwise sterilize your pipettes? Are they dedicated to PCR and PCR only? Do you sterilize and aliquot water and other such reagents? Do you have a laminar flow hood (or can you get one) to do your assays in. It can be worth the investment.
not all the time. but in our lab we have two different types of PCR and sometimes we get different results. the one which is old and have a complex operating system give good results and sharp band for multiplex PCR while the new one can not give clear and sharp band for same multiplex.
the reason behind this different results are mostly depends on ramping rate (the time taken by thermal cycler to change from one step to another).
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